WO1993007892A1 - Composition and treatment with biologically active peptides and antibiotic - Google Patents

Composition and treatment with biologically active peptides and antibiotic Download PDF

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Publication number
WO1993007892A1
WO1993007892A1 PCT/US1992/008823 US9208823W WO9307892A1 WO 1993007892 A1 WO1993007892 A1 WO 1993007892A1 US 9208823 W US9208823 W US 9208823W WO 9307892 A1 WO9307892 A1 WO 9307892A1
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WIPO (PCT)
Prior art keywords
peptide
ala
lys
seq
amino acid
Prior art date
Application number
PCT/US1992/008823
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French (fr)
Inventor
Michael Zasloff
Barry Berkowitz
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The Children's Hospital Of Philadelphia
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Publication date
Application filed by The Children's Hospital Of Philadelphia filed Critical The Children's Hospital Of Philadelphia
Priority to JP5507813A priority Critical patent/JPH07500342A/en
Publication of WO1993007892A1 publication Critical patent/WO1993007892A1/en

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1751—Bactericidal/permeability-increasing protein [BPI]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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

Definitions

  • This invention relates to biologically active peptides and proteins, and more particularly to compositions and uses
  • composition which includes at least one
  • a process wherein there is administered to a host at least one biologically active peptide or protein, said peptide or protein being an ion channel-forming peptide or protein, and an antibiotic.
  • the antibiotic in which is combined is different from such peptide or protein; i.e., such antibiotic is other than an ion channel-forming peptide or protein.
  • antibiotics examples include, but are not limited to, a tetracycline(s); a pseudomonic acid;
  • viomycin a cephalosporin; benzoyl peroxide; ethambutol;
  • Tetracyclines which may be employed include, but are not limited to, tetracycline, doxycycline, oxytetracycline, as well as those isolated from new species of Micromonospora and
  • Actimonadura such as, for example, Sch-36969, Sch-33256, and Sch-34164 (Schering Plough).
  • the pseudomonic acid is mupirocin
  • Cephalosporins which may be employed include, but are not limited to cefotaxime; cephalothin; cephalexin; cefazolin;
  • cephradine cephapirin; cefotetan; cefamandole; ceftazidime;
  • cefoxitin cefuroxime; cefoperazone; ceftriaxone; ceftizoxime; ceforanide; cefonicid; cefaclor; 3' quaternary ammonium
  • cephalosporins broad-spectrum cephalosporins such as MI-4646, MI-4648, and MI-4659 (Mochida); YM-13115 (Yamanouchi); DQ-2522 and DQ-2556 (Daiichi); BO-1232 and BO-1236 (Banyu); CL-118523 (American Cyanamid); and moxalactam.
  • the cephalosporin is cefotaxime.
  • Sulfonamides which may be employed include, but are not limited to, sulfanilamide; sulfadiazine; sulfabenzamide;
  • sulfamethoxazole sulfisoxazole; sulfamethoxazole sulfathalidine; sulfacetamide; sulfacytine; sulfadoxime; sulfamerazine;
  • sulfamethazine sulsulfamethizole
  • sulfapyridine sulfasalazine
  • sulfathiazole sulfapyrazone
  • Anti-malarial agents which may be employed include, but are not limited to, pyrimethamine; primaquine; chloroquine; quinine, and quinidine.
  • Penem antibiotics include, but are not limited to, imipenem, carbapenems, and 2-(n-azolyl) alkyl-substituted penems.
  • Aminoglycoside antibiotics include, but are not limited to, tobramycin, kanamycin, amikacin, the gentamicins (e.g.,
  • gentamicin C 1 gentamicin C 2 , gentamicin C 1a
  • netilmicin kanamycin, neomycin, streptomycin, and derivatives and analogues thereof.
  • the preferred aminoglycosides are tobramycin and the gentamicins.
  • Penicillins which may be employed in accordance with the present invention 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
  • CGP-279353 (Ciba-Geigy); an erythromycin A derivative with a cyclic carbamate fused to the C 11 /C 12 position of a macrolide ring known as A-62514 (Abbott); AC-7230 (Toyo Jozo);
  • rifamycin rifampin, carbenicillin, and nafcillin may be employed as well.
  • 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 preferred hydrophobic antibiotics are the macrolides and in particular erythromycin and derivatives and analogues thereof.
  • Peptide antibiotics which are not ion channel-forming peptides or proteins which may be employed include, but are not limited to, a bacitracin; gramacidin S; polymyxin; vancomycin; teichoplanin; and capreomycin; and derivatives and analogues thereof.
  • the biologically active amphiphilic 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. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rod-like or alpha-helical structure.
  • such peptides have at least 11 amino acids, and preferably at least 20 amino acids. In most cases, such peptides do not have in excess of 50 amino acids.
  • biologically active peptides or proteins employed in the present invention are ion channel-forming
  • An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the
  • an ion channel-forming peptide or protein is a peptide or protein which has ion
  • amphiphilic peptide or protein is a peptide which includes both hydrophobic and hydrophilic peptide regions.
  • the biologically active amphiphilic (amphipathic) ion channel-forming peptides or proteins are capable of forming ion channels, the ability of such peptides or proteins and the above-mentioned antibiotics to potentiate each other is not necessarily dependent upon the antibiotic crossing a membrane through such channels.
  • the ability to form ion channels may be a characteristic of a type of peptide or protein used in the invention, the invention is not limited to the formation and/or use of such channels as part of the mechanism for the peptide or protein potentiating the antibiotic or vice versa.
  • Applicant believes that such peptides or proteins interact with the membrane of bacterial cells and such interaction is the mechanism by which the antibiotic potentiates the peptide or protein and vice versa, the present invention is not limited to such a mechanism.
  • potentiate means either that the biologically active amphiphilic peptide or protein is effective in increasing the biological activity of the above-mentioned antibiotics against a target cell so thereby the antibiotic may be employed in an amount lower than that which would be required for preventing, destroying or inhibiting growth of a target cell, and/or that the peptide or protein may be employed in an amount lower than that which would be required for preventing,
  • the administration of the biologically active amphiphilic peptides or proteins and antibiotic to a target cell may be direct administration to the cell or systemic or topical
  • Target cells whose growth may be prevented, inhibited, or
  • Gram-positive and Gram-negative bacteria are examples of Gram-positive and Gram-negative bacteria.
  • erythromycin when employed without the above-mentioned peptides, is effective only against Gram-positive organisms. Applicants have found unexpectedly that erythromycin, when employed in combination with the above-mentioned peptides or proteins, is potentiated such that it becomes biologically effective against Gram-negative bacteria. Moreover, the
  • erythromycin may be employed against Gram-positive bacteria in amounts lower than those normally used. Furthermore, such a result can be achieved by using peptide or protein amounts lower than those normally used.
  • peptides or proteins employed in the present invention are capable of interacting selectively with membranes of
  • the peptide or protein is employed to provide peptide dosages of from 1 mg to 500 mg per kilogram of host weight, when administered systemically.
  • the peptide or protein is used in a concentration of from 0.1% to 10%.
  • 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 is generally employed in an amount of from 0.1mg to about 45mg per kg of host weight per day.
  • a combination of peptide or protein and an antibiotic such as those hereinabove described, or derivatives or analogues thereof in accordance with the present invention is effective as an antibiotic, and may be employed to inhibit, prevent or destroy the growth or proliferation of microbes, such as bacteria.
  • compositions have a broad range of potent antibiotic activity against a plurality of microorganisms, including Gram-positive and Gram-negative bacteria- Such compositions may be employed for treating or controlling microbial infection caused by organisms which are sensitive to such composition.
  • the treatment may comprise administering to a host organism or tissues acceptable to or affiliated with a microbial infection an anti-microbial amount of such peptide or protein and an
  • compositions may also be used as preservatives or sterilants for materials susceptible to microbial contamination.
  • the peptide used in conjunction with an antibiotic such as those hereinabove described, or derivatives or analogues thereof is a basic
  • 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, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids may be selected from the" class consisting of Asn, Gln, Ser, and Thr.
  • hydrophilic amino acids may be selected from the class consisting of Lys, Arg, and His, Orn, homoarginine (Har), 2,
  • Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different.
  • the polypeptide chain may comprise 5 or 6 groups of one or more of these sequences.
  • each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.
  • the polypeptide chain preferably has at least 16 amino acids, and no greater than 50 amino acids. It is to be
  • polypeptide does not have to consist entirely of the groups described above.
  • the polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention.
  • the groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary provided that in each group of the at least four groups of amino acids there are two hydrophobic and two hydrophilic amino acids as hereinabove noted.
  • the biologically active polypeptide comprises a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.
  • each of the at least four groups of amino acids which are in the peptide chain is of the sequence A-B-C-D, B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic amino acids, one of C or D is 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 2 is A-, D-A- or C-D-A-
  • Y 2 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
  • 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
  • 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.
  • the peptides may be produced by known techniques and obtained in substantially pure form. For example, the peptides may be synthesized on an automatic chemical peptide synthesizer. Journal of the American Chemical Society, Vol. 85 Pages
  • the peptide employed in conjunction with an antibiotic such as those
  • a magainin peptide is either a magainin such as Magainin I, II or III or an analogue or derivative thereof.
  • the magainin peptides may include the following basic peptide structure X 12
  • R 11 is a hydrophobic amino acid
  • R 12 is a basic hydrophilic amino acid
  • R 13 is a hydrophobic, neutral
  • R 14 and R 14a are hydrophobic or basic hydrophilic amino acids
  • R 15 is glutamic acid or aspartic acid, or a hydrophobic or basic hydrophilic amino acid
  • n is 0 or 1.
  • R 13 is a hydrophobic or neutral hydrophilic amino acid
  • R 14a is a hydrophobic or neutral hydrophilic amino acid
  • R 15 is glutamic acid or aspartic acid.
  • a magainin peptide may include the following structure:
  • a magainin peptide may also have the following structure:
  • R 16 where R 16 is a basic hydrophilic amino acid or asparagine or glutamine; or
  • R 16 -R 17 where R 17 is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid.
  • R 17 is a neutral hydrophilic amino acid.
  • a magainin peptide may also have the following structure: (Y 12 ) a - X 12 - (Z 12 ) b
  • X 12, Y 12 , and Z 12 are as previously defined, and a is 0 or 1 and b is 0 or 1.
  • the magainin peptides may also include the following basic peptide structure X 13 :
  • R 11 , R 12 , R 13 , R 14 , and R 14a are amino acids as hereinabove described.
  • the magainin peptide may also include the following
  • R 14 ) n -(R 16 ) n -(R 17 ) n - wherein R 11 , R 14 , R 14a , R 15 , R 16 , and R 17 are amino acids 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:
  • magaininin peptides refers to the basic magainin structure as well as derivatives and analogs thereof, including but not limited to the representative derivatives or analogs.
  • an antibiotic such as bacitracin, tobramycin or gentamicin or derivatives or analogues thereof 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 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:
  • R 11 is as previously defined.
  • a PGLa like peptide may also have the following structure:
  • R 11 is as previously defined.
  • a PGLa peptide may also have the following structure:
  • X 14 ; Y 14 and Z 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 16 :
  • 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:
  • R 11 and R 14 are as previously defined.
  • An XPF peptide may include the following structure:
  • An XPF peptide may also have the following structure:
  • X 16 , Y 16 and Z 16 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 sequence as given in the accompanying sequence listing:
  • CPF peptides A basic CPF peptide structure as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
  • the CPF peptide is preferably one which includes the following peptide structure X 30 :
  • R 21 is a hydrophobic amino acid
  • R 22 is a hydrophobic amino acid or a basic hydrophilic amino acid
  • R 23 is a basic hydrophilic amino acid
  • R 24 is a hydrophobic or neutral hydrophilic amino acid
  • R 25 is a basic or neutral hydrophilic amino acid.
  • the hydrophobic amino acids may be Ala, Cys, Phe, Gly, Ile, Leu, Met, Val, Trp, and Tyr.
  • the neutral hydrophilic amino acids may be Asn, Gln, Ser, and Thr.
  • the basic hydrophilic amino acids may be Lys, Arg, and 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 end 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 peptide structure may have from 1 to 4 additional amino acids at the amino end.
  • Such preferred peptides may be represented by the structural formula: Y 30 -X 30 - wherein X 30 is the hereinabove described basic peptide structure and Y 30 is
  • the carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13
  • the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:
  • X 30 is the hereinabove defined basic peptide structure
  • R 21 and R 24 are as previously defined, and R 26 is proline or a hydrophobic amino acid.
  • Preferred peptides may be represented by the following structural formula:
  • CPF peptides which may be employed in the present invention are represented by the following (single letter amino acid code):
  • CPF peptide includes the basic peptide structure as well as analogues or derivatives thereof.
  • the peptide may include one of the following basic structures X 31 through X 37 wherein:
  • X 31 is - [ R 31 -R 32 -R 32 -R 33 -R 3 1 -R 32 -R 32 ] - n ;
  • X 32 is - [R 32 -R 32 -R 33 -R 31 -R 32 -R 32 -R 3 1 ] - n ;
  • X 33 is - [R 32 -R 33 -R 31 -R 32 -R 32 -R 31 -R 32 ] - n ;
  • X 34 is - [R 33 -R 31 -R 32 -R 32 -R 31 -R 32 -R 32 ] - n ;
  • X 35 is - [R 31 -R 32 -R 32 -R 31 -R 32 -R 32 -R 33 ] - n ;
  • X 36 is - [R 32 -R 32 -R 31 -R 32 -R 32 -R 33 -R 31 ] - n ;
  • X 37 is - [R 32 -R 31 -R 32 -R 32 -R 33 -R 3 1 -R 32 ] - n ;
  • R 31 is a basic hydrophilic amino acid
  • R 32 is a
  • R 33 is a neutral hydrophilic or
  • 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),
  • Dbu 2,4-diamino-butyric acid
  • p-aminophenylalanine 2,4-diamino-butyric acid
  • the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, Ile. Leu, Met, Val, Trp and Tyr, norleucine (Nle), norvaline (Nval), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser and Thr.
  • the peptide when the peptide includes the structure X 31 , the peptide may include the following
  • Y 31 -X 31 wherein X 31 is as hereinabove described, and Y 31 is:
  • the peptide when the peptide includes the structure X 31 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide may include the following structure: X 32 - Z 32 , wl ⁇ erein X 32 is as hereinabove described, and Z 32 is:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 33 , the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 33 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 34 , the peptide may include the following structure:
  • Y 34 - X 34 , ,herein X 34 is as hereinabove described, and Y 34 is
  • the peptide when the peptide includes the structure X 34 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 35 , the peptide may include the following structure:
  • Y 35 -X 35 wherein X 35 is as hereinabove described, and Y 35 is:
  • 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 36 , the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 36 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • 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: X 37 - Z 37 wherein X 37 is as hereinabove described, and Z 37 is:
  • the peptide may include the following structure:
  • n 3
  • peptide is of one of the following structures as given in the accompanying sequence listing:
  • the biologically active amphiphilic peptide includes the following basic structure
  • R 34 is a basic hydrophilic or hydrophobic amino acid.
  • the peptide may included the following structure: Y 40 -X 40 , wherein X 40 is as hereinabove described, and Y 40 is:
  • the peptide may include the following structure:
  • X 40 -Z 40 wherein X 40 is as hereinabove described and Z 40 is:
  • the peptide has the following structural formula as given in the accompanying sequence listing:
  • the peptide has the amino acid sequence: (a)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl
  • the peptide has one of the one of the following structural formulae as given in the accompanying sequence listing:
  • the peptide may include the following structural formula:
  • n is from 2 to 5.
  • n is 3, and the peptide has the following structural formula:
  • the peptide may be selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
  • the peptide may includes the following basic structure X 50 : R 41 -R 42 -R 42 -R 41 -R 42 -R 42 -R 41 -R 41 -R 42 -R 41 -R 41 -R 41 -R 41 -R 41 -R 41 -R 41 -R 41 .
  • R 41 is a hydrophobic amino acid
  • R 42 is a basic hydrophilic or neutral hydrophilic amino acid.
  • the peptide includes the basic structure Y 50 -X 50 wherein X 50 is as hereinabove described and Y 50 is:
  • R 41 is leucine.
  • R 42 is lysine.
  • Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:
  • the includes the following basic structure X 52 :
  • R 41 is leucine. In another embodiment, R 42 is lysine.
  • the peptide includes the basic structure Y 52 -X 52 , wherein X 52 is as hereinabove described, and Y 52 is:
  • 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 52 - Z 52 , wherein X 52 is as hereinabove described, and Z 52 is :
  • the peptide may have the following structure:
  • the peptide may include the
  • the hereinabove described peptides may be acetylated with a CH 3 CO-group at the N-terminal.
  • each of the amino acid residues may be a D-amino acid residue or glycine.
  • amino acid residues may be D-amino acid or glycine residues, or L-amino acid or glycine residues.
  • the peptide employed in conjunction with an antibiotic such as those hereinabove described, or derivatives or analogues thereof is a cecropin.
  • 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 p. 108 and Christensen at al PNAS Vol. 85 p. 5072-76, which are hereby incorporated by reference.
  • cecropin includes the basic structure as well as analogues and derivatives.
  • sarcotoxins and analogues and derivatives thereof are described in Molecular Entomology, pages 369-78, in particular p. 375 Alan R. Liss Inc. (1987), which is hereby incorporated by reference.
  • sarcotoxin includes the basic materials as well as analogues and derivatives.
  • 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
  • cytolysin or pore-forming protein.
  • Defensins are described in Selsted, et al., J. Clin. Invest., Vol. 76, pgs. 1436-1439
  • 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.
  • ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as analogues or derivatives.
  • Pseudomonic acids are produced by Pseudomonas fluorescens.
  • a preferred pseudomonic acid which may be employed is mupirocin, or pseudomonic acid A, which has the following sturcture:
  • cephalosporins include a 7-aminocephalosporanic acid nucleus.
  • a preferred cephalosporin is cefotaxime, which has the following structure:
  • Ethambutol, isoniazid, and ethionamide are especially useful in treating mycobacterial infections, and in particular
  • Ethambutol is a synthetic, water soluble, heat stable compound and is the D-isomer of the structure below:
  • Ethambutol may be dispensed as the dihydrocholoride salt.
  • Ehtionamide a close chemical relative of isoniazid, has the following structure:
  • Sulfonamides have the following nucleus:
  • Penem antibiotics which may be employed include, but are not limited to, imipenem, carbapenems such as RS-533 (Sankyo), 2-(N-azolyl) alkyl-substituted penems, such as CGP- 29-718 (Ciba-Geigy), and broad-spectrum penems such as Sch- 34343 (Schering Plough).
  • Imipenem also known as a
  • thienamycin antibiotic has the following structure:
  • Preferred aminoglycoside antibiotics which may be employed are tobramhcin and the gentamicins.
  • Tobramycin has the following structure:
  • the gentamicins (Gentamicin C 1, Gentamicin C 2 , and Gentamicin C 1a ) , as well as netilmicin, have the following basic structure:
  • R 1 and R 2 are each CH 3 , the C 4 -C 5 bond is a single bond, and R 3 is H.
  • R 1 is CH 3
  • R 2 and R 3 are each H
  • the C 4 -C 5 bond is a single bond.
  • R 1a R 1, R 2 and R 3 each are H
  • the C 4 -C 5 bond is a single bond.
  • R 1 and R 2 each are H
  • R 3 is C 2 H 5
  • the C 4 -C 5 bond is a double bond.
  • aminoglycosides which may also be employed within the scope of the present invention include, but are not limited to, kanamycin and amikacin, as well as
  • metilmicin metilmicin, neomycin, and streptomycin, which is alo
  • Kanamycin and amikacin both have the following basic structure:
  • R is H
  • R is:
  • Erythromycin which is isolated from Streptomyces erythreus , is a member of a group of compounds known as macrolides.
  • the basic structure is a large lactone ring to which unusual sugars are attached.
  • macrolide refers to a large ring formed from a chain of 14 to 20 carbon atoms by lactone condensation of a carboxyl and hydroxyl group.
  • Other macrolides include oleandomycin, spiramycin, kitasamycin, and carbonmycin.
  • Erythromycin has the following structure:
  • Rokitamycin is of the following structure:
  • CGP-7040 a benzapiperazinyl rifamycin, has the following structure:
  • Preferred penicillins which may be employed in accordance with the present invention are benzyl penicillin (penicillin G) and ampicillin (alpha-amino-benzyl
  • Benzyl penicillin is of the following
  • Ampicillin has the following structure:
  • a preferred monobactam which may be employed in accordance with the present invention is aztreonam, which ha the following structure:
  • bacitracin A which has the following structure:
  • Peptide antibiotics which are not ion channel-forming peptides or proteins, which may be employed include, but are not limited to, gramacidin-S, polymyxin, vancomycin, teichoplanin, and capreomycin.
  • Vancomycin is a tricyclic glycopeptide antibiotic . Its chemical formula is C 66 H 15 Cl 2 H 9 O 24 , and it has a molecular weight of 1,449.
  • S. aureus organisms are grown to mid log phase, and then
  • MG-2 (amide) is amide-terminated Magainin II
  • Magainin II is carboxy-terminated Magainin II.
  • A-97 peptide is of the following structure:
  • 2-74 peptide is of the following structure:
  • the PGLa peptide is of the following structure:
  • the minimal inhibitory concentration (MIC) for each peptide was then measured when 20% of the minimal inhibitory
  • the gentamicin employed is a mixture of Gentamicin C 1 , Gentamicin C 1a , and Gentamicin C 2 .
  • the MIC values for bacitracin are 2 ⁇ g/ml against S. aureus, 64 ⁇ g/ml against E. coli, and >256 ⁇ g/ml against Pseudomanas aeruginosa.
  • the MIC values for gentamicin are 256 ⁇ g/ml against S . aureus, 2 ⁇ g/ml against E. coli, and >256 ⁇ g/ml against P. aeruginosa.
  • CPF Z-50 peptide is (SEQ ID NO: 21) of the CPF peptides hereinabove described.
  • Z-52 peptide is of the structure (SEQ ID NO:99)-NH 2 .
  • the minimal inhibitory concentrations are given below in Table I.
  • E. coli organisms were incubated according to the prcedure described in Example 1. After the incubation, the minimal inhibitory concentrations against E. coli of each of the peptides described in Example 1 alone, as well as of each peptide when employed in combination with 20% of the MIC for bactracin, were then measured. The results are given in Table 2 below. Table 2
  • P. aeruginosa organisms were incubated according to the procedure described in Example 1. After the incubation, the minimal inhibitory concentrations of the peptides hereinabove described alone, as well as the peptides in
  • EXAMPLE 4 In this example, E. coli or P. aeruginosa organisms were incubated according to the procedure described in Example 1.
  • the minimal inhibitory concentrations of the peptides hereinabove described alone, as well as the peptides in combination with 20% of the minimal inhibitory concentrations (MIC) of benzyl penicillin or ampicillin were then measured.
  • the MIC of benzyl penicillin against E. coli is 64 ⁇ g/ml, and of ampicillin against E. coli is 4 ⁇ g/ml. The results are given below in Table 4.
  • E. coli and P. aeruginosa organisms were incubated according to the procedure described in Example 1. After the incubation, the minimal inhibitory concentrations of the peptides hereinabove described alone, as well as the peptides in combination with 20% of the MIC of the monobactam antibiotic aztreonam, were then measured.
  • the MIC of aztreonam against E. coli is a 2 ⁇ g/ml
  • P. aeruginosa is 8 ⁇ g/ml. The results are given below in Table 5.
  • the effect of a combination of erythromycin and biologically active amphiphilic ion channel-forming peptide will be measured against K. pneumoniae, P. aeruginosa, E. coli, and S. aureus.
  • amphiphilic ion channel-forming peptide are administered to inhibit growth of Gram-negative organisms.
  • the checkerboard assay is carried out in a microtiter plate having wells arranged in rows and columns, 100 ⁇ l of plain broth is added to every row of wells. 100 ⁇ l of peptide (SEQ ID NO: 1)
  • the MIC of (SEQ ID NO:100)-NH 2 was 32 ⁇ g/ml, and the MIC of vancomycin was greater than 1,024 ⁇ g/ml; i.e., vancomycin at a concentration of 1,024 ⁇ g/ml did not inhibit growth of P. aeruginosa.
  • the following combinations of (SEQ ID NO:100)-NH 2 and vancomycin were found to be inhibitory.
  • biologically active ion channel-forming peptide are administered to inhibit growth of P. aeruginosa.
  • the peptide or protein and antibiotic such as those
  • a host is an animal, and such animal may be a human or non-human animal. It is also possible to administer the peptide or protein and antibiotic in separate forms. For example, the antibiotic may be administered systemically and the peptide or protein may be administered topically.
  • the peptide or protein and/or antibiotic such as those hereinabove described, 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.
  • the peptide or protein and/or antibiotic such as those
  • the peptide or protein When the peptide or protein is administered topically, it is 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 combination of peptide or protein and antibiotic of the present invention may be administered to a host; in particular an animal, in an effective antibiotic amount.
  • the combination when used to inhibit growth of bacterial cells, the combination, whether administered as a mixture or separately, is employed in an effective antibiotic amount.
  • antibacterial amount When used to inhibit growth of fungi, such components are administered in an effective antifungal amount.
  • 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.
  • ADDRESSEE Carella, Byrne, Bain, Gilfillan,
  • NAME/KEY Magainin II peptide.
  • NAME/KEY magainin peptide
  • NAME/KEY magainin peptide
  • NAME/KEY magainin peptide

Abstract

A composition comprising at least one biologically active amphiphilic peptide or protein, said peptide or protein being an ion channel-forming peptide or protein, and an antibiotic selected from the class consisting of non-peptide antibiotics and non-ion channel-forming peptide antibiotics, and derivatives or analogues thereof. The biologically active amphiphilic peptide or protein and the antibioic may be administered in a combined amount effective to inhibit growth of a target cell. The biologically active amphiphilic peptide or protein and antibiotic may protentiate each other.

Description

Composition and Treatment with Biologically
Active Peptides and Antibiotic
This application is a continuation-in-part of Application Serial No. 402,642, filed September 5, 19B9, which is a
continuation-in-part of Application Serial No. 339,292, filed April 17, 1989.
This invention relates to biologically active peptides and proteins, and more particularly to compositions and uses
involving biologically active peptides or proteins and an antibiotic; in particular an antibiotic which is not an ion channel-forming peptide or protein, or derivatives or analogues thereof.
It has been disclosed that agents such as polymyxin B nonapeptide, which disrupt the outer membrane of Gram-negative bacteria, increase the potency of certain antibiotics, such as fusidic acid, novobiocin, and erythromycin, against the organisms Viljanen, et al, "Susceptibility of Gram-Negative Bacteria to Polymyxin B Nonapeptide," Antimicrobial Agents and Chemotherapy. Vol. 25, No. 6, Pgs. 701-705 (June 1984). Viljanen, et al, in "Susceptibility of Gram-Negative Bacteria to the Synergistic Bactericidal Action of Serum and Polymyxin B Nonapeptide," Can. J. Microbiol.Vol. 32 pgs. 66-69 (1986), disclose a synergistic effect of polymyxin B nonapeptide and serum in bactericidal action against E.coli strains, strains of Salmonella typhimurium. Klebisiella species, Enterobacter cloacae. Pseudomonas influenzae. Vaara, et al., have disclosed a synergistic effect of polymyxin B nonapeptide and novobiocin, fusidic acid, erythromycin, clindamycin, nafcillin, and cloxacillin against smooth encapsulated E.coli and smooth Salmonella typhimurium. Antimicrobial Agents and Chemotherapy, Vol. 24, No. 1, pgs.
107-113. (July 1983).
In accordance with an aspect of the present invention, there is provided a composition which includes at least one
biologically active peptide or protein, said peptide or protein being an ion-channel forming peptide or protein; and an
antibiotic.
In accordance with another aspect of the present invention, there is provided a process wherein there is administered to a host at least one biologically active peptide or protein, said peptide or protein being an ion channel-forming peptide or protein, and an antibiotic.
The antibiotic in which is combined is different from such peptide or protein; i.e., such antibiotic is other than an ion channel-forming peptide or protein.
Although the invention is not to be limited to any
theoretical reasoning, it is believed that the peptides or proteins employed in the present invention interact with the membranes of bacterial cells and such interaction enhances the ability of the above-mentioned antibiotics to cross the membrane. It is to be understood, however, that the scope of the invention is not limited to such antibiotics.
Examples of antibiotics which may be employed include, but are not limited to, a tetracycline(s); a pseudomonic acid;
viomycin; a cephalosporin; benzoyl peroxide; ethambutol;
isoniazid; ethionamide; a sulfonamide; trimethoprim;
nitroimidazoles; an antimalariral agent; cycloserine; a penem antibiotic; an aminoglycoside; a hydrophobic antibiotic; a penicillin; a monobactam; a 50-S ribosome inhibitor; and an antibiotic having a large lipid-like lactone ring. Tetracyclines which may be employed include, but are not limited to, tetracycline, doxycycline, oxytetracycline, as well as those isolated from new species of Micromonospora and
Actimonadura, such as, for example, Sch-36969, Sch-33256, and Sch-34164 (Schering Plough).
In one embodiment, the pseudomonic acid is mupirocin
(pseudomonic acid A).
Cephalosporins which may be employed include, but are not limited to cefotaxime; cephalothin; cephalexin; cefazolin;
cephradine; cephapirin; cefotetan; cefamandole; ceftazidime;
cefoxitin; cefuroxime; cefoperazone; ceftriaxone; ceftizoxime; ceforanide; cefonicid; cefaclor; 3' quaternary ammonium
cephalosporins; broad-spectrum cephalosporins such as MI-4646, MI-4648, and MI-4659 (Mochida); YM-13115 (Yamanouchi); DQ-2522 and DQ-2556 (Daiichi); BO-1232 and BO-1236 (Banyu); CL-118523 (American Cyanamid); and moxalactam. In one embodiment, the cephalosporin is cefotaxime.
Sulfonamides which may be employed include, but are not limited to, sulfanilamide; sulfadiazine; sulfabenzamide;
sulfamethoxazole; sulfisoxazole; sulfamethoxazole sulfathalidine; sulfacetamide; sulfacytine; sulfadoxime; sulfamerazine;
sulfamethazine; sulsulfamethizole; sulfapyridine; sulfasalazine; sulfathiazole; and sulfapyrazone.
Anti-malarial agents which may be employed include, but are not limited to, pyrimethamine; primaquine; chloroquine; quinine, and quinidine.
Penem antibiotics include, but are not limited to, imipenem, carbapenems, and 2-(n-azolyl) alkyl-substituted penems.
Aminoglycoside antibiotics include, but are not limited to, tobramycin, kanamycin, amikacin, the gentamicins (e.g.,
gentamicin C1, gentamicin C2, gentamicin C1a), netilmicin, kanamycin, neomycin, streptomycin, and derivatives and analogues thereof. The preferred aminoglycosides are tobramycin and the gentamicins. The aminoglycosides, and the bacitracins
hereinafter described, tend to be hydrophilic and water-soluble.
Penicillins which may be employed in accordance with the present invention 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.
As representative examples of 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-O-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 C11/C12 position of a macrolide ring known as A-62514 (Abbott); AC-7230 (Toyo Jozo);
benzoxazinorifamycin; difficidin; dirithromycin; a
3-N-piperdinomethylzaino methyl rifamycin SV known as FCE-22250 (Farmitalia); M-119-a (Kirin Brewery); a
6-0-methyl-1-4"-O-carbamoyl erythromycin known as A-63075
(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-O-alpha-L-cladinosyl moiety, such as 3-O-alphaaL-cladinosyldeepoxy rosaramicin; tylosins and acyl demycinosyl tylosins.
In addition to the macrolides hereinabove described,
rifamycin, rifampin, carbenicillin, and nafcillin may be employed as well.
Other antibiotics which may be used (whether or not
hydrophobic) 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 preferred hydrophobic antibiotics are the macrolides and in particular erythromycin and derivatives and analogues thereof.
Peptide antibiotics which are not ion channel-forming peptides or proteins which may be employed include, but are not limited to, a bacitracin; gramacidin S; polymyxin; vancomycin; teichoplanin; and capreomycin; and derivatives and analogues thereof.
The biologically active amphiphilic peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water. In addition, the structure of such peptide provides for flexibility of the peptide molecule. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rod-like or alpha-helical structure.
In general, such peptides have at least 11 amino acids, and preferably at least 20 amino acids. In most cases, such peptides do not have in excess of 50 amino acids.
In general, the biologically active peptides or proteins employed in the present invention are ion channel-forming
peptides or proteins. 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 P. 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. As used herein an ion channel-forming peptide or protein is a peptide or protein which has ion
channel-forming properties as determined by the method of
Christensen et al. An amphiphilic peptide or protein is a peptide which includes both hydrophobic and hydrophilic peptide regions.
Although the biologically active amphiphilic (amphipathic) ion channel-forming peptides or proteins are capable of forming ion channels, the ability of such peptides or proteins and the above-mentioned antibiotics to potentiate each other is not necessarily dependent upon the antibiotic crossing a membrane through such channels. Thus, although the ability to form ion channels may be a characteristic of a type of peptide or protein used in the invention, the invention is not limited to the formation and/or use of such channels as part of the mechanism for the peptide or protein potentiating the antibiotic or vice versa. Similarly, although Applicant believes that such peptides or proteins interact with the membrane of bacterial cells and such interaction is the mechanism by which the antibiotic potentiates the peptide or protein and vice versa, the present invention is not limited to such a mechanism.
The term "potentiate", as used herein, means either that the biologically active amphiphilic peptide or protein is effective in increasing the biological activity of the above-mentioned antibiotics against a target cell so thereby the antibiotic may be employed in an amount lower than that which would be required for preventing, destroying or inhibiting growth of a target cell, and/or that the peptide or protein may be employed in an amount lower than that which would be required for preventing,
destroying, or inhibiting growth of a target cell.
The administration of the biologically active amphiphilic peptides or proteins and antibiotic to a target cell may be direct administration to the cell or 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 biologically active amphiphilic peptide or protein and antibiotic include
Gram-positive and Gram-negative bacteria.
For example, erythromycin, when employed without the above-mentioned peptides, is effective only against Gram-positive organisms. Applicants have found unexpectedly that erythromycin, when employed in combination with the above-mentioned peptides or proteins, is potentiated such that it becomes biologically effective against Gram-negative bacteria. Moreover, the
erythromycin may be employed against Gram-positive bacteria in amounts lower than those normally used. Furthermore, such a result can be achieved by using peptide or protein amounts lower than those normally used.
The peptides or proteins employed in the present invention are capable of interacting selectively with membranes of
bacteria.
In general, the peptide or protein is employed to provide peptide dosages of from 1 mg to 500 mg per kilogram of host weight, when administered systemically. When administered topically, the peptide or protein is used in a concentration of from 0.1% to 10%.
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%. When used systemically, the antibiotic or derivative or analogue thereof is generally employed in an amount of from 0.1mg to about 45mg per kg of host weight per day.
The use of a combination of peptide or protein and an antibiotic such as those hereinabove described, or derivatives or analogues thereof in accordance with the present invention is effective as an antibiotic, and may be employed to inhibit, prevent or destroy the growth or proliferation of microbes, such as bacteria.
The compositions have a broad range of potent antibiotic activity against a plurality of microorganisms, including Gram-positive and Gram-negative bacteria- Such compositions may be employed for treating or controlling microbial infection caused by organisms which are sensitive to such composition. The treatment may comprise administering to a host organism or tissues acceptable to or affiliated with a microbial infection an anti-microbial amount of such peptide or protein and an
antibiotic.
The compositions may also be used as preservatives or sterilants for materials susceptible to microbial contamination.
In accordance with a preferred embodiment, the peptide used in conjunction with an antibiotic such as those hereinabove described, or derivatives or analogues thereof 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.
In accordance with a particularly preferred embodiment, 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, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha). The neutral hydrophilic amino acids may be selected from the" class consisting of Asn, Gln, Ser, and Thr. The basic
hydrophilic amino acids may be selected from the class consisting of Lys, Arg, and 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 be the same or different. In a preferred embodiment, the polypeptide chain may comprise 5 or 6 groups of one or more of these sequences. In each group, each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.
The polypeptide chain preferably has at least 16 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. Thus, in a preferred embodiment, the biologically active polypeptide comprises a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.
In one embodiment, 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 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:
(X1)a(A-B-C-D)n(Y1)b
(X2)a(B-C-D-A)n(Y2)b
(X3)a(C-D-A-B)n(Y3)b
(X4)a(D-A-B-C)n(Y4)b wherein X1 is D; C-D- or B-C-D-, Y1 is -A or
-A-B or -A-B-C
X2 is A-, D-A- or C-D-A-
Y2 is -B, -B-C or B-C-D
X3is B-, A-B-, D-A-B-
Y3 is -C, -C-D, -C-D-A
X4is C-, B-C-, A-B-C-
Y4 is -D, -D-A, -D-A-B
a is 0 or 1; b is 0 or 1
and n is at least 4.
It is to be understood that 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.
As representative examples of peptides in accordance with the present invention, there may be mentioned.
I Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys (SEQ ID
NO:1)
II Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe- Ser-Lys. (SEQ ID NO: 2)
III Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala- (SEQ ID NO:3).
IV Ser-Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala-Phe- (SEQ ID NO: 4)
V Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser (SEQ ID NO: 5)
The peptide may have amino acids extending from either end of the chain. For example, 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.
Similarly, in any polypeptide chain having at least four groups of amino acids of the sequence as described above, the chain may have, for example, a C-D sequence before the first A-B-C-D group. Also other amino acid sequences may be attached to the "A" and/or the "D" end of one of these polypeptide chains. Also there may be amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other. The peptides may be produced by known techniques and obtained in substantially pure form. For example, the peptides may be synthesized on an automatic chemical peptide synthesizer. Journal of the American Chemical Society, Vol. 85 Pages
2149-54(1963). It is also possible to produce such peptides by recombinant DNA technology using genetic engineering techniques. Methods in Enzymology, Vol. 68: "Recombinant DNA" (Wu, et al.) Academic Press, New York (1979); and Methods in Enzymology, Vol. 185: "Gene Expression Technology" (D.V. Goeddel, et al.) Academic Press, New York (1990).
In accordance with another preferred embodiment, the peptide employed in conjunction with an antibiotic such as those
hereinabove described, or derivatives or analogues thereof 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 peptides may include the following basic peptide structure X12
╌ R11-R11-R12-R13-R11-R14-R12-R11-R14-
R12-R11-R11-R11-R14a- (R15 )n-R14a-R14 ╌
wherein R11 is a hydrophobic amino acid, R12 is a basic hydrophilic amino acid; R13 is a hydrophobic, neutral
hydrophilic, or basic hydrophilic amino acid; R14 and R14a are hydrophobic or basic hydrophilic amino acids, R15 is glutamic acid or aspartic acid, or a hydrophobic or basic hydrophilic amino acid, and n is 0 or 1. In a preferred embodiment, R13 is a hydrophobic or neutral hydrophilic amino acid, R14a is a
hydrophobic amino acid, and R15 is glutamic acid or aspartic acid.
Thus, for example, a magainin peptide may include the following structure:
Y12 - X12,
where X12 is the hereinabove described basic peptide
structure and Y12 is
(i) R12; (ii) R14a - R12;
(iii) R11-R14a-R12; or
(iv) R14-R11-R14a-R12
where R11, R12, R14, and R14a are as previously defined. A magainin peptide may also have the following structure:
-X12-Z12,
wherein X12 is as previously defined and Z12 is:
(i) R16 where R16 is a basic hydrophilic amino acid or asparagine or glutamine; or
(ii) R16-R17 where R17 is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid. Preferably, R17 is a neutral hydrophilic amino acid.
A magainin peptide may also have the following structure: (Y12)a - X12 - (Z12)b
where X12, Y12 , and Z12 are as previously defined, and a is 0 or 1 and b is 0 or 1.
The magainin peptides may also include the following basic peptide structure X13:
R14-R11-R14a-R12-R11-R11-R12-R13-R11-R14-
R12-R11-R11-R12-,
wherein R11, R12, R13, R14, and R14a are amino acids as hereinabove described.
The magainin peptide may also include the following
structure X13-Z13; wherein X13 is the hereinabove described basic peptide structure and Z13 is
(R11)n-(R11)n-(R11)n-(R14a)n-(R15)n-(R14a)n-
(R14)n-(R16)n-(R17)n-, wherein R11, R14, R14a, R15, R16, and R17 are amino acids 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.
As representative examples of such magainin peptides, there may be mentioned peptides having the following primary sequences as given in the accompanying sequence listing, as well as appropriate analogues and derivatives thereof:
(a) (NH2) (SEQ ID NO: 6) (OH) or (NH2)
(Magainin I)
(b) (NH2) (SEQ ID NO: 7) (OH) or (NH2)
(Magainin II)
(c) (NH2) (SEQ ID NO:8) (OH) or (NH2)
(Magainin III)
The following are examples of peptide derivatives or analogs of the basic structure:
(d) (NH2) (SEQ ID NO: 9) (OH) or (NH2)
(e) (NH2) (SEQ ID NO:10) (OH) or (NH2 )
(f) (NH2) (SEQ ID NO: 11) (OH) or (NH2)
Magainin peptides are described in Proc. Natl. Acad Sci.
Vol. 84, pp. 5449-53 (Aug. 1987). 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.
In accordance with a further embodiment, the peptide
employed in conjunction with an antibiotic such as bacitracin, tobramycin or gentamicin or derivatives or analogues thereof 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 X14:
- R11-R17-R12-R11-R14-R11-R11-
R11-R14-R12-R11-R11-R12-R11-
R11-R11-R12- where R11, R12, R14, and R17 are as previously defined. The PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl end.
Thus, for example, a PGLa peptide may have the following structure:
Y14-X14,
where X14 is as previously defined and
Y14 is
(i) R11,
(ii) R14-R11
where R11 is as previously defined.
For example, a PGLa like peptide may also have the following structure:
X14-Z14,
where X14 is as previously defined ; and Z 14 is :
( i ) R1 1 ; or
( ii ) R11-R11
where R11 is as previously defined.
A PGLa peptide may also have the following structure:
(Y14)a-X14-(Z14) b
where X14; Y14 and Z14 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 X16:
—R11-R17-R12-R11-R14-R18-R17-
R11-R14-R12-R11-R11-R12-
R11-R11- R11-R12-R15-R11-,
wherein R11 R12, R14, R15 and R17 are as previously defined and R18 is glutamine or asparagine. 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.
Thus, for example, an XPF peptide may include the following structure:
Y16-X16-,
where X16 is as previously defined and Y16 is
(i) R11 or
(ii) R14-R11
where R11 and R14 are is as previously defined.
An XPF peptide may include the following structure:
-X16-Z16,
where X16 is as previously defined and Z16 is
(i) R11; or
(ii) R11-R18; or
(iii) R11-R18-Proline; or
(iv) R11-R18-Proline-R12
An XPF peptide may also have the following structure:
(Y16)a -X16(Z16)b
where X16, Y16 and Z16 are as previously defined: a is 0 or 1 and b is 0 or 1.
Preferred are XPF or PGLa peptides, which are characterized by the following primary amino acid sequence as given in the accompanying sequence listing:
PGLa : (SEQ ID NO: 12) (NH2)
XPF : (SEQ ID NO: 13)
A review of XPF and PGLa can be found in Hoffman et al, EMBOJ.2:711-714, 1983; Andreu et al, J. Biochem. 149:531-535, 1985; Gibson et al J. Biol. Chem. 261:5341-5349, 1986; and Giovannini et al, Biochem J. 243:113-120, 1987. In accordance with yet another embodiment, the peptide employed in conjunction with an antibiotic such as those
hereinabove described, or derivatives or analogues thereof may be a CPF peptide or appropriate analogue or derviative thereof.
A basic CPF peptide structure as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
The CPF peptide is preferably one which includes the following peptide structure X30:
-R21-R21-R22-R22-R21-R21-R23-R21-
-R21-R21-R23-R21-R21-R24-R25-R21- wherein R21 is a hydrophobic amino acid;
R22 is a hydrophobic amino acid or a basic hydrophilic amino acid;
R23 is a basic hydrophilic amino acid; and
R24 is a hydrophobic or neutral hydrophilic amino acid; and
R25 is a basic or neutral hydrophilic amino acid.
The hereinabove basic structure is hereinafter symbolically indicated as X30.
The hydrophobic amino acids may be Ala, Cys, Phe, Gly, Ile, Leu, Met, Val, Trp, and Tyr.
The neutral hydrophilic amino acids may be Asn, Gln, Ser, and Thr.
The basic hydrophilic amino acids may be Lys, Arg, and 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 end 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 peptide structure may have from 1 to 4 additional amino acids at the amino end.
Accordingly, such preferred peptides may be represented by the structural formula: Y30-X30- wherein X30 is the hereinabove described basic peptide structure and Y30 is
(i) R25-, or
(ii) R22-R25; or
(iii) R21-R22-R25; or
(iv) R22-R21-R22-R25; Preferably
Glycine -R21-R22-R25- wherein R21, R22, and R25 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.
In a preferred embodiment, the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:
-X30-Z30 wherein
X30 is the hereinabove defined basic peptide structure and
Z30 is
(i) R21-,
(ii) R21-R21-;
(iii) R21-R21-R24;
(iv) R21-R21-R24-R24;
(v) R21-R21-R24_R24-R26;
(vi) R21-R21-R24-R24-R26-Gln; or
(vii) R21-R21-R24-R24-R26-Gln-Gln,
wherein R21 and R24 are as previously defined, and R26 is proline or a hydrophobic amino acid.
Preferred peptides may be represented by the following structural formula:
(Y30)a -X30-(Z30) b
wherein X30, Y30 and Z30 are as previously defined and a is 0 or 1 and b is 0 or 1. Representative examples of CPF peptides which are useful in the present invention have been described in the literature and comprise 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 NO: 21)
(SEQ ID NO:22)
(SEQ ID NO: 23)
(SEQ ID NO:24)
(SEQ ID NO: 25)
(SEQ ID NO: 26)
A review of the CPF peptides can be found in Richter, K., Egger, R., and Kreil (1986) J. Biol. Chem. 261, 3676-3680;
Wakabayashi, T. Kato, H., and Tachibaba, S. (1985) Nucleic Acids Research 13, 1817-1828; Gibson, B.W., Poulter, L., Williams, D.H., and Maggio, J.E. (1986) J. Biol. Chem. 261, 5341-5349.
CPF peptides which may be employed in the present invention are represented by the following (single letter amino acid code):
G12S3LG4ALKA5LKIG678LGG9(10)QQ
Where:
1 = F , L
2 = G, A
3 = F , L
4 = K, L
5 = A , G, T
6 = A, T
7 = H, N
8 = A , M, F , 9 = A, S , T
10 P, L
The numbered amino acids may be employed as described in any combination to provide either a basic CPF peptide structure or an analogue or derivative. The term CPF peptide includes the basic peptide structure as well as analogues or derivatives thereof.
In accordance with yet another embodiment, the peptide may include one of the following basic structures X31 through X37 wherein:
X31 is - [ R31-R32-R32-R33-R3 1-R32-R32 ] -n;
X32 is - [R32-R32-R33-R31-R32-R32-R3 1 ] -n;
X33 is - [R32-R33-R31-R32-R32-R31-R32 ] -n;
X 34 is - [R33-R31-R32-R32-R31-R32-R32 ] -n;
X35 is - [R31-R32-R32-R31-R32-R32-R33 ] -n;
X36 is - [R32-R32-R31-R32-R32-R33 -R31 ] -n; and
X37 is - [R32-R31-R32-R32-R33-R3 1-R32] -n;
wherein R31 is a basic hydrophilic amino acid, R32 is a
hydrophobic amino acid, R33 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-diamino-butyric acid (Dbu), and p-aminophenylalanine.
The hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, Ile. Leu, Met, Val, Trp and Tyr, norleucine (Nle), norvaline (Nval), and cyclohexylalanine (Cha).
The neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser and Thr.
In accordance with one embodiment, when the peptide includes the structure X31, the peptide may include the following
structure:
Y31-X31, wherein X31 is as hereinabove described, and Y31 is:
(i) R32; (ii) R32-R32;
(iii) R31-R32-R32;
(iv) R33-R31-R32-R32;
(V) R32-R33-R31-R32-R32; or
(vi) R32-R32-R33-R31-R32-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X31, the peptide may include the following structure:
X31-Z31, wherein X31 is as hereinabove described, and Z31 is:
(i) R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R33;
(V) R31-R32-R32-R33-R31; or
(vi) R31-R32-R32-R33-R31-R32.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y31)a-X31-(Z31)b, wherein Y31 and Z31 are as previously defined, a is 0 or 1, and b is 0 or 1.
When the peptide includes the structure X32, the peptide may include the following structure:
Y32 - X32, wherein X32 is as hereinabove described, and Y32 is:
( i ) R31 ;
( ii) R32-R31;
( iii ) R32-R32-R31 ;
( iv) R31-R32-R32-R31 ;
(V) R33-R31-R32-R32-R31; or
(vi) R32-R33-R31-R32-R32-R31.
In another embodiment, when the peptide includes the
structure X32, the peptide may include the following structure: X32 - Z32, wlαerein X32 is as hereinabove described, and Z32 is:
( i ) R32 ;
(ii ) R32-R32 ;
(iii ) R32-R32-R33 ;
(iv) R32-R32-R33-R31;
(v) R32-R32-R33-R31-R32 ; or
(vi ) R32-R32-R33-R31-R32-R32 .
In accordance with yet another embodiment, the peptide may include the following structure:
(Y32)a - X32 - (Z32)b, wherein Y32 and Z32 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with another embodiment, when the peptide includes the structure X33, the peptide may include the following structure:
Y33 - X33 wherein X33 is as hereinabove described, and Y33 is:
(i) R32;
(ii) R31-R32;
(iii) R32-R31-R32;
(iv) R32-R32-R31-R32;
(v) R31-R32-R32-R31--R32; or
(vi) R33-R31-R32-R32-R31-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X33, the peptide may include the following structure:
X33 - Z33 wherein X33 is as hereinabove described, and Z33 is:
(i) R32;
(ii) R32-R33;
(iii) R32-R33-R31;
(iv) R32-R33-R31-R32;
(v) R32-R33-R31-R32-R32; or (vi ) R32-R33-R31-R32-R32-R31 .
In accordance with yet another embodiment, the peptide may include the following structure:
(Y33)a - X33 - (Z33)b, wherein Y33 and Z33 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with yet another embodiment, when the peptide includes the structure X34, the peptide may include the following structure:
Y34 - X34, ,herein X34 is as hereinabove described, and Y34 is
(i) R32;
(ii) R32-R32;
(iii) R31-R32-R32;
(iv) R3 Y2-R31-R32-R32;
(v) R32-R32-R31-R32-R32; or
(vi) R31-R32-R32-R31-R32-R32, wherein R31, R32 and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X34, the peptide may include the following structure:
X34-Z34, wherein X34 is as hereinabove described, and Z34 is:
(i) R33;
(ii) R33-R31;
(iii) R33-R31-R32;
(iv) R33-R31-R32-R32;
(v) R33-R31-R32-R32-R31; or
(vi) R33-R31-R32-R32-R31-R32.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y34)a- X34- (Z34)b, wherein X34 and Z34 are as previously defined, a is 0 or 1, and b is 0 or 1. In accordance with a further embodiment, when the peptide includes the structure X35, the peptide may include the following structure:
Y35-X35, wherein X35 is as hereinabove described, and Y35 is:
(i) R33;
(ii) R32-R33;
(iii) R32-R32-R33;
(iv) R31-R32-R32-R33;
(v) R32-R31-R32-R32-R33; or
(vi) R32-R32-R31-R32-R32-R33, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X35, the peptide may include the following structure:
X35 - Z35 wherein X35 is as hereinabove described, and Z35 is:
(i) R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R31;
(V) R31-R32-R32-R31-R32; or
(vi) R31-R32-R32-R31-R32-R32.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y35) - X35 (Z35)b, wherein X35 and Z35 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with a further embodiment, when the peptide includes the structure X36, the peptide may include the following structure:
Y36 - X36 wherein X36 is as hereinabove described, and Y36 is:
(i) R31;
(ii) R33-R31; (iii) R32-R33-R31;
(iv) R32-R32-R33-R31;
(V) R31-R32-R32-R33-R31; or
(vi) R32-R31-R32-R32-R33-R31, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X36, the peptide may include the following structure:
X36-Z36, wherein X36 is as hereinabove described, and Z36 is:
(i) R32;
(ii) R32-R32;
(iii) R32-R32-R31;
(iv) R32-R32-R31"R32;
(v) R32-R32-R31-R32-R32; or
(vi) R32-R32-R31-R32-R32-R33.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y36) - X36 (Z36)b, wherein Y36 and Z36 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with one embodiment, when the peptide includes the structure X37, the peptide may includes the structure
Y37-X37, wherein X37 is as hereinabove described, and Y37 is:
(i) R32;
(ii) R31-R32;
(iii) R33-R31-R32;
(iv) R32-R33-R31-R32;
(v) R32-R32-R33-R31-R32; or
(vi) R31-R32-R32-R33-R31-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with a further embodiment, when the peptide includes the structure X37, the peptide may include the following structure: X37 - Z37 wherein X37 is as hereinabove described, and Z37 is:
(i) R32;
(ii) R32-R31;
(iii) R32-R31-R32;
(iv) R32-R31-R32-R32;
(v) R32-R3l-R32-R32-R33; or
(vi) R32-R3l-R32-R32-R33-R31.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y37) - X37 (Z37)b, wherein Y37 and Z37 are as previously defined, a is 0 or 1, and b is 0 or 1.
In a preferred embodiment, n is 3, and most preferably the peptide is of one of the following structures as given in the accompanying sequence listing:
(Lys Ile Ala Gly Lys Ile Ala)3 (SEQ ID NO:27).
(Lys Ile Ala Lys Ile Ala Gly)3 (SEQ ID NO:28).
(Lys Ile Ala Gly Lys Ile Gly)3 (SEQ ID NO:29).
(Lys Leu Ala Gly Lys Leu Ala)3 (SEQ ID NO:30).
(Lys Phe Ala Gly Lys Phe Ala)3 (SEQ ID NO: 31).
(Lys Ala Leu Ser Lys Ala Leu)3 (SEQ ID NO:32).
(Lys Leu Leu Lys Ala Leu Gly)3 (SEQ ID NO:33).
(Lys Ala Ile Gly Lys Ala Ile)3 (SEQ ID NO:34).
(Gly Ile Ala Lys Ile Ala Lys)3 (SEQ ID NO:35).
(Lys Ile Ala Lys Ile Phe Gly)3 (SEQ ID NO:36).
(Gly Ile Ala Arg Ile Ala Lys)3 (SEQ ID NO:37).
(Lys Phe Ala Arg Ile Ala Gly)3 (SEQ ID NO:38).
(Gly Phe Ala Lys Ile Ala Lys)3 (SEQ ID NO:39).
(Lys Ile Ala Gly Orn Ile Ala)3 (SEQ ID NO:40).
(Lys Ile Ala Arg Ile Ala Gly)3 (SEQ ID NO:41).
(Orn Ile Ala Gly Lys Ile Ala)3 (SEQ ID NO:42).
(Gly Ile Ala Arg Ile Phe Lys)3 (SEQ ID NO: 43).
(Lys Nle Ala Gly Lys Nle Ala)3 (SEQ ID NO: 44).
(Lys Nle Ala Gly Lys Ile Ala)3 (SEQ ID NO:45). ( Lys Ile Ala Gly Lys Nle Ala ) 3 ( SEQ ID NO : 46 ) .
( Lys Nva Ala Gly Lys Nva Ala ) 3 ( SEQ ID NO : 47 ) .
( Lys Nva Ala Gly Lys Ile Ala) 3 ( SEQ ID NO: 48 ) .
(Lys Leu Leu Ser Lys Leu Gly)3 (SEQ ID NO: 49).
(Lys Leu Leu Ser Lys Phe Gly)3 (SEQ ID NO: 50).
(Lys Ile Ala Gly Lys Nva Ala)3 (SEQ ID NO:51).
(His Ile Ala Gly His Ile Ala)3 (SEQ ID NO:52).
(Ala Gly Lys Ile Ala Lys Ile)3 (SEQ ID NO:53).
(Ile Ala Lys Ile Ala Gly Lys)3 (SEQ ID NO:54).
(Lys Ile Ala Gly Arg Ile Ala)3 (SEQ ID NO:55).
(Arg Ile Ala Gly Arg Ile Ala)3 (SEQ ID NO:56).
(Lys Val Ala Gly Lys Ile Ala)3 (SEQ ID NO:57).
(Lys Ile Ala Gly Lys Val Ala)3 (SEQ ID NO:58).
(Ala Lys Ile Ala Gly Lys Ile)3 (SEQ ID NO:59).
(Orn Ile Ala Gly Orn Ile Ala)3 (SEQ ID NO:60).
(Lys Phe Ala Gly Lys Ile Ala)3 (SEQ ID NO: 61).
(Lys Ile Ala Gly Lys Phe Ala)3 (SEQ ID NO:62).
(Lys Cha Ala Gly Lys Ile Ala)3 (SEQ ID NO: 63).
(Lys Nle Ala Lys Ile Ala Gly)3 (SEQ ID NO: 64).
(Arg Ile Ala Gly Lys Ile Ala)3 (SEQ ID NO:65).
(Har Ile Ala Gly Har Ile Ala)3 (SEQ ID NO:66).
(Xaa Ile Ala Gly Lys Ile Ala)3 (SEQ ID NO:67).
(Lys Ile Ala Gly Xaa Ile Ala)3 (SEQ ID NO:68).
In (SEQ ID NO: 67) and (SEQ ID NO:68), Xaa is
p-aminophenylalanine.
In accordance with another embodiment, the biologically active amphiphilic peptide includes the following basic structure
X40:
R31-R32-R32-R33-R34-R32-R32-R31-R32-R32-R32-R34-R32-R32, wherein R31, R32, and R33 are as hereinabove described, a
R34 is a basic hydrophilic or hydrophobic amino acid.
In accordance with one embodiment, the peptide may includ the following structure: Y40-X40, wherein X40 is as hereinabove described, and Y40 is:
(i) R32;
(ii) R32-R32;
(iii) R34-R32-R32;
(iv) R33-R34-R32-R32;
(v) R32-R33-R34-R32-R32;
(v) R32-R32-R33-R34-R32-R32, or
(vii) R31-R32-R32-R33-R34-R32-R32,wherein R31, R32, R33 and R 34 are as hereinabove described.
In accordance with another embodiment, the peptide may include the following structure:
X40-Z40, wherein X40 is as hereinabove described and Z40 is:
(i) R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R33;
(v) R31-R32-R32-R33-R34;
(vi) R31-R32-R32-R33-R34-R32 ; or
(vii) R31-R32-R32-R33-R34-R32-R32, wherein R31, R32, R33, and R34 are as hereinabove described.
In accordance with yet another embodiment the peptide may include the following structure:
(Y40)a-X40-(Z40)b, wherein Y40 and Z40 are as Previously defined, a is 0 or 1, and b is 0 or 1. In a preferred
embodiment, the peptide has the following structural formula as given in the accompanying sequence listing:
(SEQ ID NO: 69)
In another preferred embodiment, the peptide has the
following structural formula as given in the accompanying
sequence listing:
(SEQ ID NO:70) In accordance with a further embodiment, the peptide has one of the one of the following structural formulae as given in the accompanying sequence listing:
(SEQ ID NO:71)
(SEQ ID NO: 72)
(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 77)
(SEQ ID NO: 78)
(SEQ ID NO: 79)
(SEQ ID NO: 80)
(SEQ ID NO: 81)
(SEQ ID NO: 82)
(SEQ ID NO: 83)
(SEQ ID NO: 84)
(SEQ ID NO: 85)
In accordance with another embodiment, the peptide may include the following structural formula:
- (Lys Ile Ala Lys Lys Ile Ala)-n, wherein n is from 2 to 5. Preferably, n is 3, and the peptide has the following structural formula:
(Lys Ile Ala Lys Lys Ile Ala)3
(SEQ ID NO: 86)
In accordance with another embodiment, the peptide may be selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
(SEQ ID NO: 87)
(SEQ ID NO: 88)
(SEQ ID NO: 89)
(SEQ ID NO: 90)
In accordance with another embodiment, the peptide may includes the following basic structure X50: R41-R42-R42-R41-R42-R42-R41-R41-R42-R41-R41.
R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
In one embodiment, the peptide includes the basic structure Y50-X50 wherein X50 is as hereinabove described and Y50 is:
(i) R41;
{ii) R42-R41; or
(iii) R42-R42-R41, wherein R41 and R42 are as hereinabove described.
In one embodiment, R41 is leucine. In another embodiment, R42 is lysine. Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:
(SEQ ID NO: 91)
( SEQ ID NO : 92 )
(SEQ ID NO : 93 )
(SEQ ID NO : 94)
In accordance with another embodiment, the includes the following basic structure X52 :
R42-R41-R42-R42-R41-R41-R42-R42-R41-R42-R42, wherein R41 is a hydrophobic amino acid, and R2 is a basic hydrophilic or neutral hydrophilic amino acid.
In one embodiment R41 is leucine. In another embodiment, R42 is lysine.
In one embodiment, the peptide includes the basic structure Y52-X52, wherein X52 is as hereinabove described, and Y52 is:
(i) R42;
(ii) R41-R42;
(iii) R41-R41-R42;
(iv) R42-R41-R41-R42; or
(v) R42-R42-R41-R41-R42.
In one embodiment, the peptide may have the following structure: Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu
5 10
Leu Lys Lys Leu Arg Arg
15
(SEQ ID NO. :95)
In another embodiment, the peptide includes the basic structure X52 - Z52, wherein X52 is as hereinabove described, and Z52 is :
( i ) R41;
( ii) R41-R41;
( iii ) R4l-R41-R42;
( iv) R41-R41-R42-R42; or
(v ) R41-R41-R42-R42-R41;
In one embodiment, the peptide may have the following structure:
Lys Leu Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys Lys Leu
5 10 15
(SEQ ID NO: 96)
In another embodiment, the peptide may include the
structure:
(Y52)a - X52 - (Z52)b, wherein X52, Y52 and Z52 are as hereinabove described, and a is 0 or 1, and b is 0 or 1.
In one embodiment, the hereinabove described peptides may be acetylated with a CH3CO-group at the N-terminal.
In accordance with another embodiment, each of the amino acid residues may be a D-amino acid residue or glycine.
Although the scope of this particular embodiment is not to be limited to any theoretical reasoning, it is believed that the peptides and proteins of the present invention, when consisting entirely of D-amino acid or glycine residues, have increased resistance to proteolytic enzymes while retaining their
biological activity. Such peptides may thus be administered orally. Also, in accordance with another embodiment, all of the amino acid residues may be D-amino acid or glycine residues, or L-amino acid or glycine residues.
In still another embodiment, the peptide employed in conjunction with an antibiotic such as those hereinabove described, or derivatives or analogues thereof is a cecropin.
The term 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 p. 108 and Christensen at al PNAS Vol. 85 p. 5072-76, which are hereby incorporated by reference.
The term cecropin includes the basic structure as well as analogues and derivatives.
In yet another embodiment, the peptide employed in
conjunction with an antibiotic such as those hereinabove
described, or derivatives or analogues thereof is a sarcotoxin. The sarcotoxins and analogues and derivatives thereof are described in Molecular Entomology, pages 369-78, in particular p. 375 Alan R. Liss Inc. (1987), which is hereby incorporated by reference.
The term sarcotoxin includes the basic materials as well as analogues and derivatives.
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. 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 (1981). 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.
The term ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as analogues or derivatives.
Pseudomonic acids are produced by Pseudomonas fluorescens. A preferred pseudomonic acid which may be employed is mupirocin, or pseudomonic acid A, which has the following sturcture:
Figure imgf000035_0001
The cephalosporins include a 7-aminocephalosporanic acid nucleus. A preferred cephalosporin is cefotaxime, which has the following structure:
Figure imgf000035_0002
Ethambutol, isoniazid, and ethionamide are especially useful in treating mycobacterial infections, and in particular
infections of M. tuberculosis. Ethambutol is a synthetic, water soluble, heat stable compound and is the D-isomer of the structure below:
Figure imgf000036_0004
Ethambutol may be dispensed as the dihydrocholoride salt. Ehtionamide, a close chemical relative of isoniazid, has the following structure:
Figure imgf000036_0003
Sulfonamides have the following nucleus:
Figure imgf000036_0002
to which various R radical in the amido group (-SO2NHR) have been attached or in which various substitutions of the amino group (NH2) are made. Representative examples of the
sulfonamides and their structure are given hereinbelow:
Figure imgf000036_0001
Figure imgf000037_0001
Penem antibiotics which may be employed include, but are not limited to, imipenem, carbapenems such as RS-533 (Sankyo), 2-(N-azolyl) alkyl-substituted penems, such as CGP- 29-718 (Ciba-Geigy), and broad-spectrum penems such as Sch- 34343 (Schering Plough). Imipenem, also known as a
thienamycin antibiotic, has the following structure:
Figure imgf000037_0002
Preferred aminoglycoside antibiotics which may be employed are tobramhcin and the gentamicins. Tobramycin has the following structure:
Figure imgf000038_0003
The gentamicins (Gentamicin C1, Gentamicin C2, and Gentamicin C1a) , as well as netilmicin, have the following basic structure:
Figure imgf000038_0002
For Gentamicin C1, R1 and R2 are each CH3, the C4-C5 bond is a single bond, and R3 is H. For Gentamcin C2, R1 is CH3, R2 and R3 are each H, and the C4-C5 bond is a single bond. For Gentamicin C1a, R1, R2 and R3 each are H, and the C4-C5 bond is a single bond. For netilmicin, R1 and R2 each are H, R3 is C2H5, and the C4-C5 bond is a double bond.
Other aminoglycosides which may also be employed within the scope of the present invention include, but are not limited to, kanamycin and amikacin, as well as
metilmicin, neomycin, and streptomycin, which is alo
effective in treating tuberculosis. Kanamycin and amikacin both have the following basic structure:
Figure imgf000038_0001
For kanamycin, R is H, and for amikacin, R is:
Figure imgf000039_0001
Erythromycin, which is isolated from Streptomyces erythreus , is a member of a group of compounds known as macrolides. The basic structure of a group of compounds known as macrolides. The basic structure is a large lactone ring to which unusual sugars are attached. The term
"macrolide" refers to a large ring formed from a chain of 14 to 20 carbon atoms by lactone condensation of a carboxyl and hydroxyl group. Other macrolides include oleandomycin, spiramycin, kitasamycin, and carbonmycin.
Erythromycin has the the following structure:
Figure imgf000039_0002
It is also to be understood that other macrolide antibiotics, such as roxythromycin, clarithromycin, and others hereinabove described, may be employed as well.
For purposes of illustration of examples of other macrolide structures, the following examples, in addition to the above structure of erythromycin, are given below.
Rokitamycin is of the following structure:
Figure imgf000040_0001
CGP-7040, a benzapiperazinyl rifamycin, has the following structure:
Figure imgf000040_0002
It is to be understood, however, that the scope of the present invention is not to be limited to the specific macrolides or macrolide structures hereinabove described.
Preferred penicillins which may be employed in accordance with the present invention are benzyl penicillin (penicillin G) and ampicillin (alpha-amino-benzyl
penicillin). Benzyl penicillin is of the following
structure:
Figure imgf000041_0001
Ampicillin has the following structure:
Figure imgf000041_0002
A preferred monobactam which may be employed in accordance with the present invention is aztreonam, which ha the following structure:
Figure imgf000041_0003
The bacitracins which may be employed in accordance with the present invention tend to be hydrophilic and water soluble. The preferred bacitracin is bacitracin A, which has the following structure:
Figure imgf000042_0001
Peptide antibiotics, which are not ion channel-forming peptides or proteins, which may be employed include, but are not limited to, gramacidin-S, polymyxin, vancomycin, teichoplanin, and capreomycin.
Vancomycin is a tricyclic glycopeptide antibiotic . Its chemical formula is C66H15Cl2H9O24, and it has a molecular weight of 1,449.
It is to be understood, however, that the scope of the invention is not to be limited to the specific antibiotics and antibiotic structures hereinabove described.
The present invention will be further described with respect to the following examples, however, the scope of the invention is not to be limited thereby.
Example 1
S. aureus organisms are grown to mid log phase, and then
3
diluted to 10 organisms/ml in 1/2 strength trypticase soy broth.
After the incubation of the organisms, the minimal inhibitory concentraion in ug/ml for Z-52 MG-2 (amide-terminated), PGLa, CPF
Z-50, A-97, Magainin II, and Z-74 peptides, against S. aureus was measured when each peptide was added alone to the organisms.
"MG-2 (amide)" is amide-terminated Magainin II, and "Magainin II" is carboxy-terminated Magainin II. A-97 peptide is of the following structure:
(SEQ ID NO: 97)-amide
2-74 peptide is of the following structure:
(SEQ ID NO: 98)-amide
The PGLa peptide is of the following structure:
(SEQ ID NO: 12)-amide.
The minimal inhibitory concentration (MIC) for each peptide was then measured when 20% of the minimal inhibitory
concentration of bacitracin, tobramycin or gentamicin,
respecitvely, is added to the organisms along with the peptide. The gentamicin employed is a mixture of Gentamicin C1, Gentamicin C1a, and Gentamicin C2. For Examples 1-3, the MIC values for bacitracin are 2 μg/ml against S. aureus, 64 μg/ml against E. coli, and >256 μg/ml against Pseudomanas aeruginosa. The MIC values for gentamicin are 256 μg/ml against S . aureus, 2 μg/ml against E. coli, and >256 μg/ml against P. aeruginosa. The MIC values for tobramycin are >256 μg/ml against S. aureus, 2 μg/ml against E. coli, and 128 μg/ml against P. aeruginosa. CPF Z-50 peptide is (SEQ ID NO: 21) of the CPF peptides hereinabove described. Z-52 peptide is of the structure (SEQ ID NO:99)-NH2. The minimal inhibitory concentrations are given below in Table I.
TABLE I
MIC With
Peptide Added Antibiotic
Peptide Alone Bacitracin Gentamicin Tobramycin
Z-52 32 8 16 16
MG-2 amide 256 32 4 32
PGLa 32 16 2 32
A-97 32 16 4 32
Magainin 2 256 128 4 32
Z-50 16 16 2 8
Z-74 16 16 4 8
EXAMPLE 2
E. coli organisms were incubated according to the prcedure described in Example 1. After the incubation, the minimal inhibitory concentrations against E. coli of each of the peptides described in Example 1 alone, as well as of each peptide when employed in combination with 20% of the MIC for bactracin, were then measured. The results are given in Table 2 below. Table 2
Peptide MIC
Peptide Peptide with
Alone Bacitracin
Z-52 8 8
MG-2 amide 16 4
PGLa 16 8
Z-50 16 16
A-97 8 4
Magainin II 32 8
(amide)
Z-74 16 8
Example 3
In this example, P. aeruginosa organisms were incubated according to the procedure described in Example 1. After the incubation, the minimal inhibitory concentrations of the peptides hereinabove described alone, as well as the peptides in
combination with 20% of the MIC of bacitracin, were then
measured. The results are given below in Table 3.
Table 3
Peptide MIC
Peptide Peptide with
Alone Bacitracin Added
Z-52 16 2
MG-2 amide 128 8
PGLa 64 8
Z-50 32 4
A-97 32 4
Magainin II 256 16
Z-74 16 16
EXAMPLE 4 In this example, E. coli or P. aeruginosa organisms were incubated according to the procedure described in Example 1.
After, the incubation, the minimal inhibitory concentrations of the peptides hereinabove described alone, as well as the peptides in combination with 20% of the minimal inhibitory concentrations (MIC) of benzyl penicillin or ampicillin, were then measured. The MIC of benzyl penicillin against E. coli is 64 μg/ml, and of ampicillin against E. coli is 4 μg/ml. The results are given below in Table 4.
TABLE 4
E. coli-
MIC
Peptide Peptide and Peptide and
Peptide alone Benzyl penicillin Ampicillin
Z-52 16 16
Mg 2-amide 64 32 8
PGLa 32 16
Magainin
II 64 32 16
Z-50 32 8
A-97 32 16 16
Z-74 4 8
P. aeruginosa-
Peptide
PGLa 128 32 32
Mg-2
amide 256 128 128 EXAMPLE 5
E. coli and P. aeruginosa organisms were incubated according to the procedure described in Example 1. After the incubation, the minimal inhibitory concentrations of the peptides hereinabove described alone, as well as the peptides in combination with 20% of the MIC of the monobactam antibiotic aztreonam, were then measured. The MIC of aztreonam against E. coli is a 2 μg/ml, and against P. aeruginosa is 8 μg/ml. The results are given below in Table 5.
TABLE 5
E. coli-
MIC
Peptide Peptide with
Peptide alone Aztreonam
Z-52 16 2
Mg 2-amide 64 32
PGLa 32 4
Z-50 32 8
A-97 32 8
Magainin
II 64 16
Z-74 4 8
P. aeruginosa-
Peptide
Z-52 32 32
Mg-2
amide 128 64
PGLa 128 64
Z-50 64 32
A-97 32 32
Magainin
II 256 128
Z-74 32 8
The above results indicate that when one of the biologically active ion-channel forming peptides hereinabove described is added in combination with bacitracin, tobramycin, gentamicin,. benzyl penicillin, ampicillin, or aztreonam, in an amount of 20% of the MIC of these antibiotics, against S. aureus, E.coli, or P. aeruginosa, there is, in most cases, a resulting synergy between the peptide and the antibiotic. In most cases, less peptide and less antibiotic may be used against these organisms when peptide and antibiotic are employed in combination, than if peptide or antibiotic alone were employed.
Example 6
In this example, the effect of a combination of erythromycin and biologically active amphiphilic ion channel-forming peptide will be measured against K. pneumoniae, P. aeruginosa, E. coli, and S. aureus.
For each of the organisms listed in Table 6 below, 10 organisms were mid-log inoculated into 200 μl of one-half strength trypticase soy broth. The organisms were incubated for 15 hrs. at 37°C. After the incubation of the organisms, the minimal inhibitory concentration in μg/ml for Magainin II
(amide-terminated), shown as MGN2, CPF Z-50 (amide-terminated) peptide, Z-52 (amide-terminated) peptide, against each species of organism was measured wherein 10 μg/ml of erythromycin was added and wherein no erythromycin was added. The minimal inhibitory concentration of erythromycin alone against each species of organism was also measured. The minimal inhibitory
concentrations in μg/ml are given below in Table 6. For purposes of explanation, the "+" and "-" signs below the "Erythromycin (10 μg/ml)" column indicate the presence or absence, respectively, of erythromycin administered in combination with one of the biologically active peptides. K.pneumoniae, P. aeruginosa, and E. coli are Gram-negative bacteria.
Table 6
Minimal Inhibitory
Concentration (μg/ml)
Erthro- MGN2 CPF Z-52 Erytho mycin
Organism (10μg/ml) amide z-50 amide mycin amide
K.Pneumoniae - 5 <0.5 1.5 >100
+ <0.5 <0.5 <0.5
P.aeruαuinosa - 50-100 <5.0 5-15 >100
+ <5.0 <0.5 <1.5
E.coli - 5 <5.0 1.5 >100
+ 0.5 <0.5 <0.5
The above results show that when one of the biologically active ion channel-forming peptides shown in Table 6 is added to 10 μg/ml of erythromycin, such a
combination of peptide and erythromycin is effective against the Gram-negative organisms shown, whereas greater than 100 μg/ml of erythromycin alone is required for effective biological activity against these Gram-negative organisms. The addition of erythromycin to the biologically active peptides also enables one to use less of the biologically active peptide against Gram-negative organisms. Thus, there is provided a synergistic effect against Gram-negative organisms when erythromycin and a biologically active
amphiphilic ion channel-forming peptide are administered to inhibit growth of Gram-negative organisms.
It was also found that a concentration from about 0.6 μg/ml to about 1.25 μg/ml of erythromycin alone was required for effective biological activity against S. aureus, a Gram-positive organism, and that greater than 250 μg/ml of amide-terminated organism, and that greater than 250 μg/ml of amide-terminated Magainin II alone was required for effective biological activity against S.aureus. A combination of 10 μg/ml of amide-terminated Magainin II and 0.03 ug/ml of erythromycin, however, also showed effective biological activity against S. aureus. Thus, it has also been shown that an enhanced effect against Gram-positive organisms is also obtained when erythromycin and a biologically active amphiphilic ion channel-forming peptide are administered to inhibit growth of Gram-positive organisms. The addition of erythromycin to the biologically active peptide enables one to use less of the biologically active peptide against a
Gram-positive organism.
Example 7
The procedure for this assay, which is a checkerboard assay, is performed as described in Antibiotics and Laboratory Medicine, 2 nd ed., Victor Lorian, M.D., Editor, pgs. 540-546 (1986).
The checkerboard assay is carried out in a microtiter plate having wells arranged in rows and columns, 100 μl of plain broth is added to every row of wells. 100 μl of peptide (SEQ ID
NO: 100) at 512 μg/ml is added to the top row of wells, and serially diluted (1:2) through the columns. 50μl of vancomycin in 4X concentrations is added to each appropriate column of wells to give a range of vancomycin concentrations from 16 to 1,024 μg/ml. 50μl of P. aeruginosa strain ATCC 27853 is then added to each well. The plate is incubated at 35°-37°C for 18 to 24 hours, and the wells are then examined for the presence of visible growth, i.e., turbidity.
Peptide (SEQ ID NO:100)-NH2 was tested alone or in
combination with vancomycin for activity against P. aeruginosa strain ATCC 27853.
In this assay, the MIC of (SEQ ID NO:100)-NH2 was 32 μg/ml, and the MIC of vancomycin was greater than 1,024 μg/ml; i.e., vancomycin at a concentration of 1,024 μg/ml did not inhibit growth of P. aeruginosa. The following combinations of (SEQ ID NO:100)-NH2 and vancomycin were found to be inhibitory.
8 μg/ml (SEQ ID NO:100)-NH2 plus 16 μg/ml vancomycin
8 μg/ml (SEQ ID NO:100)-NH2 plus 32 μg/ml vancomycin
8 μg/ml (SEQ ID NO:100)-NH2 plus 64 μg/ml vancomycin
8 μg/ml (SEQ ID NO:100)-NH2 plus 128 μg/ml vancomycin
8 μg/ml (SEQ ID NO:100)-NH2 plus 256 μg/ml vancomycin
8 μg/ml (SEQ ID NO:100)-NH2 plus 512 μg/ml vancomycin
8 μg/ml (SEQ ID NO:100)-NH2 plus 1,024 μg/ml vancomycin
The above results thus indicate that an enhanced effect against P. aeruginosa is obtained when vancomycin and a
biologically active ion channel-forming peptide are administered to inhibit growth of P. aeruginosa.
The peptide or protein and antibiotic such as those
hereinabove described, may be employed for treating a wide variety of hosts. In accordance with a preferred embodiment, a host is an animal, and such animal may be a human or non-human animal. It is also possible to administer the peptide or protein and antibiotic in separate forms. For example, the antibiotic may be administered systemically and the peptide or protein may be administered topically.
The peptide or protein and/or antibiotic such as those hereinabove described, 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. 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 and/or antibiotic such as those
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. When the peptide or protein is administered topically, it is 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. Examples of 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 combination of peptide or protein and antibiotic of the present invention may be administered to a host; in particular an animal, in an effective antibiotic amount. When used to inhibit growth of bacterial cells, the combination, whether administered as a mixture or separately, is employed in an effective
antibacterial amount. When used to inhibit growth of fungi, such components are administered in an effective antifungal amount.
As representative examples of administering the peptide or protein and antibiotic for topical or local administration, 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.
Numerous modifications and variations of the present
invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.
SEQUENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT: Berkowitz, Barry A.
Zasloff, Michael
(ii) TITLE OF INVENTION: Composition and Treatment with Biologically Active Peptides and Antibiotic.
(iii) NUMBER OF SEQUENCES: 100
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Carella, Byrne, Bain, Gilfillan,
Cecchi & Stewart
(B) STREET: 6 Becker Farm Road
(C) CITY: Roseland
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07068
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch diskette
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: DW4.V2
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER:
US07402642
(B) FILING DATE: 05-SEP-1989
(A) APPLICATION NUMBER:
US07339292
(B) FILING DATE: 17-APR-1989
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Olstein, Elliot M.
(B) REGISTRATION NUMBER: 24,025
(C) REFERENCE/DOCKET NUMBER: 421250
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Ala Phe Ser Lys Ala Phe Ser Lys Ala Phe
5 10
Ser Lys Ala Phe Ser Lys Ala Phe Ser Lys
15 20
(2) INFORMATION FOR SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989
(xi) SEQUENCE DESCPIPTION: SEQ ID NO: 2 : Ala Phe Ser Lys Ala Phe Ser Lys Ala Phe
5 10
Ser Lys Ala Phe Ser Lys Ala Phe Ser Lys
15 20
Ala Phe Ser Lys
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Phe Ser Lys Ala Phe Ser Lys Ala Phe Ser
5 10
Lys Ala Phe Ser Lys Ala
15
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Ser Lys Ala Phe Ser Lys Ala Phe Ser Lys
5 10
Ala Phe Ser Lys Ala Phe Ser Lys Ala Phe
15 20
(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Lys Ala Phe Ser Lys Ala Phe Ser Lys Ala
5 10
Phe Ser Lys Ala Phe Ser
15
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(A) NAME/KEY: Magainin I peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy of Sciences
(D) VOLUME: 84 (F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6
Gly Ile Gly Lys Phe Leu His Ser Ala Gly
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu Ile
15 20
Met Lys Ser
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Magainin II peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987 (J) PUBLICATION DATE: 07-MAR-1989
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7
Gly Ile Gly Lys Phe Leu His Ser Ala Lys
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu Ile
15 20
Met Asn Ser
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Magainin III peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8
Gly Ile Gly Lys Phe Leu His Ser Ala Lys
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu Ile
15 20
Met Asn
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: magainin peptide.
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C ) JOURNAL : Proceedings of the National Academy of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Ile Gly Lys Phe Leu His Ser Ala Lys Lys
5 10
Phe Gly Lys Ala Phe Val Gly Glu Ile Met
15 20
Asn Ser
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: magainin peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Gly Lys Phe Leu His Ser Ala Lys Lys Phe
5 10
Gly Lys Ala Phe Val Gly Glu Ile Met Asn
15 20
Ser
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: magainin peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Lys Phe Leu His Ser Ala Lys Lys Phe Gly
5 10
Lys Ala Phe Val Gly Glu Ile Met Asn Ser
15 20
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: PGLa peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Hoffman, et al.
(C) JOURNAL: EMBO J.
(D) VOLUME: 2
(F) PAGES: 711-714
(G) DATE: 1983
(A) AUTHOR: Andreu, et al.
(C) JOURNAL: Journal of Biochemistry
(D) VOLUME: 149
(F) PAGES: 531-535
(G) DATE: 1985
(A) AUTHOR: Gibson, et al.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986 (A) AUTHOR: Giovannini, et al.
(C) JOURNAL: Biochem J.
(D) VOLUME: 243
(F) PAGES: 113-120
(G) DATE: 1987
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Gly Met Ala Ser Lys Ala Gly Ala Ile Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: XPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Hoffman, et al. l
(C) JOURNAL: EMBO J.
(D) VOLUME: 2
(F) PAGES: 711-714
(G) DATE: 1983
(A) AUTHOR: Andreu, et al .
(C) JOURNAL: Journal of Biochemistry
(D) VOLUME: 149 (F) PAGES: 531-535
(G) DATE: 1985
(A) AUTHOR: Gibson, et al.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(A) AUTHOR: Giovannini, et al.
(C) JOURNAL: Biochem J.
(D) VOLUME: 243
(F) PAGES : 113-120
(G) DATE : 1987
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Gly Trp Ala Ser Lys Ile Gly Gln Thr Leu
5 10
Gly Lys Ile Ala Lys Val Gly Leu Lys Glu
15 20
Leu Ile Gln Pro Lys
25
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION: (A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL : J. Biol. Chem.
(D) VOLUME : 261
(F) PAGES : 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME : 261
(F) PAGES : 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Gly Phe Gly Ser Phe Leu Gly Leu Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ala Pro Gln Gln
25 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson,. B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349 (G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Gly Leu Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Gly Leu Lys Ile Gly Ala His Leu
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME : 261
(F) PAGES : 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Gly Leu Ala Ser Leu Leu Gly Lys Ala Leu
5 10
Lys Ala Gly Leu Lys Ile Gly Thr His Phe
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE; peptide (ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407 (I) FILING DATE: 16-OCT-1989 (J) PUBLICATION DATE: 03-MAY-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Gly Leu Ala Ser Leu Leu Gly Lys Ala Leu
5 10
Lys Ala Thr Leu Lys Ile Gly Thr His Phe
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828 (G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Met
15 20
Leu Gly Gly Thr Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION: (A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMEER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ala Pro Gln Gln
25 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349 (G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ser Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T. Kato, H.
Tachibaba, S.
(C) JOURNAL : Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Leu
15 20
Leu Gly Gly Thr Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE; 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ala Pro Gln Gln
25 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C ) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349 (G) DATE : 1986
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Met
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME; 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research (D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys He Gly Ala Asn Ala
15 20
Leu Gly Gly Ser Leu Gln Gln
25
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K. Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Gly Leu Lys Ile Gly Thr Asn Phe
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE :
(A) NAME/KEY: CPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME : 261
(F) PAGES : 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES : 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES : 5341-5349
(G) DATE: 1986 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Gly Leu Ala Ser Leu Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ser Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii ) MOLECULE TYPE : peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Lys Ile Ala Lys Ile Ala Gly Lys Ile Ala
5 10
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: Lys Ile Ala Gly Lys Ile Gly Lys Ile Ala
5 10
Gly Lys Ile Gly Lys He Ala Gly Lys Ile
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: Lys Leu Ala Gly Lys Leu Ala Lys Leu Ala
5 10
Gly Lys Leu Ala Lys Leu Ala Gly Lys Leu
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Lys Phe Ala Gly Lys Phe Ala Lys Phe Ala
5 10
Gly Lys Phe Ala Lys Phe Ala Gly Lys Phe
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Lys Ala Leu Ser Lys Ala Leu Lys Ala Leu
5 10
Ser Lys Ala Leu Lys Ala Leu Ser Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Lys Leu Leu Lys Ala Leu Gly Lys Leu Leu
5 10
Lys Ala Leu Gly yys Leu Leu Lys Ala Leu
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS;
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE : peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: Lys Ala Ile Gly Lys Ala Ile Lys Ala Ile
5 10
Gly Lys Ala Ile Lys Ala Ile Gly Lys Ala
15 20
Ile
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE; amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUECCE DESCRIPTION: SEQ ID NO:35: Gly Ile Ala Lys Ile Ala Lys Gly Ile Ala
5 10
Lys Ile Ala Lys Gly Ile Ala Lys Ile Ala
15 20
Lys
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Lys Ile Ala Lys Ile Phe Gly Lys Ile Ala
5 10
Lys Ile Phe Gly Lys Ile Ala Lys Ile Phe
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Gly Ile Ala Arg Ile Ala Lys Gly Ile Ala
5 10
Arg He Ala Lys Gly Ile Ala Arg Ile Ala
15 20
Lys
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Lys Phe Ala Arg Ile Ala Gly Lys Phe Ala
5 10
Arg Ile Ala Gly Lys Phe Ala Arg Ile Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY; linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Gly Phe Ala Lys Ile Ala Lys Gly Phe Ala
5 10
Lys Ile Ala Lys Gly Phe Ala Lys Ile Ala
15 20
Lys
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
( ii) MOLECULE TYPE : peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: Lys Ile Ala Gly Orn Ile Ala Lys Ile Ala
5 10
Gly Orn Ile Ala Lys Ile Ala Gly Orn Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Lys Ile Ala Arg Ile Ala Gly Lys He Ala
5 10
Arg He Ala Gly Lys Ile Ala Arg He Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: Orn Ile Ala Gly Lys Ile Ala Orn Ile Ala
5 10
Gly Lys Ile Ala Orn Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: Gly Ile Ala Arg Ile Phe Lys Gly Ile Ala
5 10
Arg Ile Phe Lys Gly Ile Ala Arg Ile Phe
15 20
Lys
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: Lys Nle Ala Gly Lys Nle Ala Lys Nle Ala
5 10
Gly Lys Nle Ala Lys Nle Ala Gly Lys Nle
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: Lys Nle Ala Gly Lys Ile Ala Lys Nle Ala
5 10
Gly Lys Ile Ala Lys Nle Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: Lys Ile Ala Gly Lys Nle Ala Lys Ile Ala
5 10
Gly Lys Nle Ala Lys Ile Ala Gly Lys Nle
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: Lys Nva Ala Gly Lys Nva Ala Lys Nva Ala
5 10
Gly Lys Nva Ala Lys Nva Ala Gly Lys Nva
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Lys Nva Ala Gly Lys Ile Ala Lys Nva Ala
5 10
Gly Lys Ile Ala Lys Nva Ala Gly Lys Nva
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: Lys Leu Leu Ser Lys Leu Gly Lys Leu Leu
5 10
Ser Lys Leu Gly Lys Leu Leu Ser Lys Leu
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH; 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: Lys Leu Leu Ser Lys Phe Gly Lys Leu Leu
5 10
Ser Lys Phe Gly Lys Leu Leu Ser Lys Phe
15 20
Gly
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Lys Ile Ala Gly Lys Nva Ala Lys Ile Ala
5 10
Gly Lys Nva Ala Lys Ile Ala Gly Lys Nva
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: His Ile Ala Gly His He Ala His Ile Ala
5 10
Gly His Ile Ala His Ile Ala Gly His Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Ala Gly Lys Ile Ala Lys Ile Ala Gly Lys
5 10
He Ala Lys Ile Ala Gly Lys He Ala Lys
15 20
Ile
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: Ile Ala Lys Ile Ala Gly Lys Ile Ala Lys
5 10
Ile Ala Gly Lys Ile Ala Lys Ile Ala Gly
15 20
Lys
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: Lys Ile Ala Gly Arg Ile Ala Lys Ile Ala
5 10
Gly Arg Ile Ala Lys Ile Ala Gly Arg Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY; linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Arg Ile Ala Gly Arg Ile Ala Arg Ile Ala
5 10
Gly Arg Ile Ala Arg Ile Ala Gly Arg Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Lys Val Ala Gly Lys Ile Ala Lys Val Ala
5 10
Gly Lys Ile Ala Lys Val Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: Lys Ile Ala Gly Lys Val Ala Lys Ile Ala
5 10
Gly Lys Val Ala Lys Ile Ala Gly Lys Val
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH; 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE; peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: Ala Lys Ile Ala Gly Lys Ile Ala Lys Ile
5 10
Ala Gly Lys Ile Ala Lys Ile Ala Gly Lys
15 20
Ile
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYP:: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: Orn Ile Ala Gly Orn Ile Ala Orn Ile Ala
5 10
Gly Orn Ile Ala Orn Ile Ala Gly Orn Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: Lys Phe Ala Gly Lys Ile Ala Lys Phe Ala
5 10
Gly Lys Ile Ala Lys Phe Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62
Lys Ile Ala Gly Lys Phe Ala Lys Ile Ala
5 10
Gly Lys Phe Ala Lys Ile Ala Gly Lys Phe
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 63;
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: Lys Cha Ala Gly Lys Ile Ala Lys Cha Ala
5 10
Gly Lys Ile Ala Lys Cha Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: Lys Nle Ala Lys Ile Ala Gly Lys Nle Ala
5 10
Lys Ile Ala Gly Lys Nle Ala Lys Ile Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 AMINO ACIDS
(B) TYPE: amino acids
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: Arg Ile Ala Gly Lys Ile Ala Arg He Ala
5 10
Gly Lys Ile Ala Arg Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: Har Ile Ala Gly Har Ile Ala Har Ile Ala
5 10
Gly Har Ile Ala Har Ile Ala Gly Har Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE: Xaa is p-aminophenylalanine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: Xaa Ile Ala Gly Lys Ile Ala Xaa Ile Ala
5 10
Gly Lys Ile Ala Xaa Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE: Xaa is p-aminophenylalanine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: Lys Ile Ala Gly Xaa Ile Ala Lys Ile Ala
5 10
Gly Xaa Ile Ala Lys Ile Ala Gly Xaa Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: Lys Leu Ala Ser Lys Ala Gly Lys Ile Ala Gly
5 10
Lys Ile Ala Lys Val Ala Leu Lys Ala Leu
15 20
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION; SEQ ID NO: 70: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala Gly
5 10
Orn Ile Ala Lys Ile Ala Gly Lys Ile Ala
15 20
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Arg Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Nle Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Nva He Ala Lys Ile Ala Gly Lys He
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: Lys Phe Ala Gly Lys Phe Ala Lys Phe Ala Gly
5 10
Orn Phe Ala Lys Phe Ala Gly Lys Phe Ala
15 20
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: Lys Ile Ala Gly Lys Phe Ala Lys Ile Ala
5 10
Gly Orn Phe Ala Lss Ile Ala Gly Lys Phe
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: Lys Ile Ala Gly Lys Nle Ala Lys Ile Ala
5 10
Gly Orn Nle Ala Lys Ile Ala Gly Lys Nle
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: Lys Met Ala Ser Lys Ala Gly Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78 Lys Ile Ala Ser Lys Ala Gly Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala Leu
15 20
(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE; amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: Lys Ile Ala Ser Lys Ala Gly Lys Nle Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala Leu
15 20
(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Lys Leu Ala Ser Lys Ala Gly Lys Nle Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
Lys Nle Ala Ser Lys Ala Gly Lys Nle Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala Leu
15 20
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is p-aminophenylalanine. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Xaa Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: Lys Ile Ala Gly Ala Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Ala Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Ala Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: Lys Ile Ala Lys Lys Ile Ala Lys Ile Ala
5 10
Lys Lys Ile Ala Lys Ile Ala Lys Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS;
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: Ala Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88; Lys Ile Ala Gly Lys Ile Ala Ala Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Ala Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
Gly Met Ala Ser Lys Ala Gly Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
Leu Lys Lys Leu Lys Lys Leu Leu Lys Leu
5 10
Leu
(2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY; linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys
5 10
Leu Leu
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu
5 10
Lys Leu Leu
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION;SEQ ID NO: 94:
Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu
5 10
Leu Lys Leu Leu
(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at
N-terminus.
(ix) SEQUENCE DESCRIPTION: SEQ ID NO:95:
Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys Lys Leu Arg Arg
5 10 15
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
Lys Leu Lys Lys Leu Leu Lys Lys Leu Lys
5 10
Lys Leu Leu Lys Leu Leu
15
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
Gly Ile Gly Lys Phe Leu His Ser Ala Gly
5 10
Lys Phe Gly Lys Ala Phe Val Lys Ile Met
15 20
Lys Ser (2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: Ala Gly Ile Gly Lys Phe Leu His Ala Ala
5 10
Lys Lys Phe Ala Lys Ala Phe Val Ala Glu
15 20
Ile Met Asn Ser
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: Ala Leu Ser Lys Ala Leu Ser Lys Ala Leu
5 10
Ser Lys Ala Leu Ser Lys Ala Leu Ser Lys
15 20
Ala Leu Ser Lys
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100
Gly Ile Lys Lys Phe Leu Lys Lys Ala Gly
5 10
Lys Phe Gly Lys Ala Phe
15

Claims

WHAT IS CLAIMED IS
1. A process of inhibiting growth of a target cell in a 3ost, comprising:
administering to a host at least one biologically active amphiphilic peptide or protein, said peptide or protein being an ion channel-forming peptide or protein; and
an antibiotic which is not an ion channel-forming peptide or protein, said biologically active amphiphilic peptide or protein and said antibiotic being administered in a combined amount effective to inhibit growth of a target cell in a host.
2. The process of Claim 1 wherein said antibiotic is selected from the class consisting of a tetracycline; a
pseudomonic acid; benzoyl peroxide; neomycin; viomycin; a cephalosporin; rifampin; ethambutol; streptomycin; isoniazid; ethionamide; a sulfonaide; trimethoprin; cycloserine; a
nitroimidazole; and a penem antibiotic.
3. The process of Claim 2 wherein said antibiotic is a cephalosporin.
4. The process of Claim 3 wherein said cephalosporin is selected from the group consisting of cephalothin, cephalexin, cefazolin, cephradine, cephapirin, cefamandole, cefoxitin, cefoperazone, cefotaxime, moxalactam, and 3' quaternary ammonium cephalosporins.
5. The process of Claim 4 wherein the cephalosporin is cefotaxime.
6. The process of Claim 2 wherein said antibiotic is a tetracycline.
7. The process of Claim 2 wherein said antibiotic is a sulfonamide.
8. The process of Claim 7 wherein said sulfonamide is selected from the class consisting of sulfanilamide,
sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfamethoxazole, and sulfathalidine.
9. The process of Claim 1 wherein the peptide is a basic polypeptide having at least sixteen amino acids, wherein said basic polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids.
10. The process of Claim 1 wherein the peptide is a magainin peptide.
11. The process of Claim 1 wherein the peptide is an XPF peptide.
12. The process of Claim 1 wherein the peptide is a PGLa peptide.
13. The process of Claim 1 wherein the peptide is a CPF peptide.
14. The process of Claim 1 wherein the peptide includes one of the following basic structures X31 through X37' wherein:
X31 is -[R31-R32-R32-R33-R31-R32-R32]n-;
X32 is -[R32-R32-R33-R31-R32-R32-R31]n-;
X33 is -[R32-R33-R31-R32-R32-R31-R32]n-;
X34 is -[R33-R31-R32-R32-R31-R32-R32]n-;
X35 is -[R31-R32-R32-R31-R32-R32-R33]n-;
X36 is -[R32-R32-R31-R32-R32-R33-R31]n-; and
X37 is -[R32-R31-R32-R32-R33-R31-R32]n-, wherien R31 is a basic hydrophilic amino acid, R33 is a hydrophobic amino acid,
R33 is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5.
15. The process of Claim 1 wherein the peptide includes the following basic structure X40:
R31-R32-R32-R33-R34-R32-R32-R31-R32-R32-R32-R34-R32-R32, wherein
R31 is a basic hydrophilic amino acid, R32 is a hydrophobic amino acid, and R33 is a neutral hydrophilic, hydrophobic or basic hydrophilic amino acid, and R34 is a basic hydrophilic or
hydrophobic amino acid.
16. The process of Claim 1 wherein the peptide includes.the following basic structure X50:
R41-R42-R42-R41-R42-R42-R41-R41-R42-R41-R41, wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
17. The process of Claim 1 wherein the peptide includes the following basic structure X52:
R42-R41-R42-R42-R41 R41-R42-R42-R41-R42-R42,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
18. A composition comprising:
(a) at least one biologically active amphiphilic peptide or protein, said peptide or protein being an ion
channel-forming peptide or protein; and
(b) an antibiotic which is not an ion channel-forming peptide or protein, and derivatives and analogues thereof.
19. The composition of Claim 18 wherein said components (a) and (b) are present in a combined amount effective to inhibit growth of a target cell.
20. The composition of Claim 19 wherein said antibiotic is selected from the class consisting of a tetracycline; a
pseudomonic acid; benzoyl peroxide; neomycin; viomycin; a
cephalosporin; rifampin; ethambutol; streptomycin; isoniazid;
ethionamide; a sulfomamide; trimethoprim; cycloserine; a
nitroimidazole, and a penem antibiotic.
21. The composition of Claim 20 wherein the non-peptide antibiotic is a cephalosporin.
22. The compositon of Claim 21 wherein said cephalosporin is selected from the group consisting of cephalothin, cephalexin, cefazolin, cephradine, cephapirin, cefamandole, cefoxitin, cefoperazone, cefotaxime, moxalactam, and 3' quaternary ammonium cephalosporins.
23. The composition of Claim 22 wherein the cephalosporin is cefotaxime.
24. The composition of Claim 20 wherein said antibiotic. is a tetracycline.
25. The composition of Claim 20 wherein said antibiotic is a sulfonamide.
26. The composition of Claim 25 wherein said sulfonamide is selected from the class consisting of sulfanilamide,
sulfadiazine, sulfamethoxazole, sulfisoxazole, and
sulfathalidine.
27. The composition of Claim 18 wherein the peptide is a basic polypeptide having at least sixteen amino acids, wherein said basic polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids.
28. The composition of Claim 18 wherein the peptide is a magainin peptide.
29. The composition of Claim 18 wherein the peptide is an XPF peptide.
30. The composition of Claim 18 wherein the peptide is a PGLa peptide.
31. The composition of Claim 18 wherein the peptide is a CPF peptide.
32. The composition of Claim 18 wherein the peptide includes one of the following basic structures X31 through X37' wherein:
X31 is -[R31-R32-R32-R33-R31-R32-R32]n-;
X32 is -[R32-R32-R33-R31-R32-R32-R31]n-;
X33 is -[R32-R33-R31-R32-R32-R31-R32]n-;
X34 is -[R33-R31-R32-R32-R31-R32-R32]n-;
X35 is -[R31-R32-R32-R31-R32-R32-R33]n-;
X36 is -[R32-R32-R31-R32-R32-R33-R31]n-; and
X37 is -[R32-R31-R32-R32-R 33-R31-R32]n-; wherein R3, is a basic hydrophilic amino acid, R32 is a hydrophobic amino acid, R33 is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5.
33. The composition of Claim 18 wherein the peptide
includes the following basic structure X40: R31-R32-R32-R33-R34-R32-R32-R31-R32-R32-R32-R34-R32-R32, wherein R31 is a basic hydrophilic amino acid, R32 is a hydrophobic amino acid, and R33 is a neutral hydrophilic, hydrophobic, or basic hydrophilic amino acid, and R34 is a basic hydrophilic or hydrophobic amino acid.
34. The composition of Claim 18 wherein the peptide includes the following basic structure X50:
R41-R42-R42-R41-R42-R42-R41-R4l-R42-R41-R41,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
35. The composition of Claim 18 wherein the peptide includes the following basic structure X52:
R42-R41-R42-R42-R41-R41-R42-R42-R41-R42-R42,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
36. The process of Claim 1 wherein each of said peptide and said antibiotic is administered in an amount ineffective in inhibiting growth of a target cell in a host if administered alone to a host.
37. The process of Claim 1 wherein the peptide includes the following structural formula:
-(Lys Ile Ala Lys Lys Ile Ala)n-, wherein n is from 2 to 5.
38. The composition of Claim 18 wherein the peptide
includes the following structural formula:
-(Lys Ile Ala Lys Lys Ile Ala)n-, wherein n is from 2to 5.
PCT/US1992/008823 1991-10-16 1992-10-16 Composition and treatment with biologically active peptides and antibiotic WO1993007892A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP5507813A JPH07500342A (en) 1991-10-16 1992-10-16 Compositions having biologically active peptides and antibiotics and treatments using the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US77877191A 1991-10-16 1991-10-16
US778,771 1991-10-16

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Publication number Priority date Publication date Assignee Title
FR2715847A1 (en) * 1994-02-08 1995-08-11 Rhone Poulenc Rorer Sa Composition containing nucleic acids, preparation and uses
EP0563844B1 (en) * 1992-03-30 1999-02-24 Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) Antimicrobial compositions and pharmaceutical preparations thereof
CN114432428A (en) * 2022-02-24 2022-05-06 西北农林科技大学 Application of PGLa in improving sensitivity of bacteria to antibiotics and delaying generation of drug resistance of bacteria

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US4617149A (en) * 1983-09-21 1986-10-14 Eli Lilly And Company Growth hormone release factor analogs
US4636489A (en) * 1983-07-07 1987-01-13 Plantorganwerk Kg Modified protease inhibitors, process for their preparation, and pharmaceutical compositions prepared therefrom
US4659692A (en) * 1982-11-19 1987-04-21 The Regents Of The University Of California Cationic oligopeptides having microbicidal activity
US4668662A (en) * 1984-04-18 1987-05-26 Hoechst Aktiengesellschaft Polypeptides with an anticoagulant action, a process to prepare or obtain them, their use and agents containing them
US4791100A (en) * 1985-07-17 1988-12-13 Hoechst Aktiengesellschaft Novel polypeptides with a blood coagulation-inhibiting action, processes for their preparation and isolation, their use and agents containing them

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Publication number Priority date Publication date Assignee Title
US4107298A (en) * 1976-04-29 1978-08-15 Ab Bonnierforetagen Antigenically active polypeptide and a process for its preparation
US4659692A (en) * 1982-11-19 1987-04-21 The Regents Of The University Of California Cationic oligopeptides having microbicidal activity
US4636489A (en) * 1983-07-07 1987-01-13 Plantorganwerk Kg Modified protease inhibitors, process for their preparation, and pharmaceutical compositions prepared therefrom
US4617149A (en) * 1983-09-21 1986-10-14 Eli Lilly And Company Growth hormone release factor analogs
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US4791100A (en) * 1985-07-17 1988-12-13 Hoechst Aktiengesellschaft Novel polypeptides with a blood coagulation-inhibiting action, processes for their preparation and isolation, their use and agents containing them

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Publication number Priority date Publication date Assignee Title
EP0563844B1 (en) * 1992-03-30 1999-02-24 Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) Antimicrobial compositions and pharmaceutical preparations thereof
FR2715847A1 (en) * 1994-02-08 1995-08-11 Rhone Poulenc Rorer Sa Composition containing nucleic acids, preparation and uses
WO1995021931A1 (en) * 1994-02-08 1995-08-17 Rhone-Poulenc Rorer S.A. Nucleic acid-containing composition, its preparation and use
CN114432428A (en) * 2022-02-24 2022-05-06 西北农林科技大学 Application of PGLa in improving sensitivity of bacteria to antibiotics and delaying generation of drug resistance of bacteria

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CA2120337A1 (en) 1993-04-17
JPH07500342A (en) 1995-01-12
AU2901692A (en) 1993-05-21

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