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WO1993011783A1 - Composition de peptides biologiquement actifs et d'agents de chelation et procede de traitement associe - Google Patents

Composition de peptides biologiquement actifs et d'agents de chelation et procede de traitement associe Download PDF

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Publication number
WO1993011783A1
WO1993011783A1 PCT/US1992/010427 US9210427W WO9311783A1 WO 1993011783 A1 WO1993011783 A1 WO 1993011783A1 US 9210427 W US9210427 W US 9210427W WO 9311783 A1 WO9311783 A1 WO 9311783A1
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WO
WIPO (PCT)
Prior art keywords
peptide
ala
amino acids
basic
lys
Prior art date
Application number
PCT/US1992/010427
Other languages
English (en)
Inventor
Barry Berkowitz
Original Assignee
Magainin Pharmaceuticals Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Magainin Pharmaceuticals Inc. filed Critical Magainin Pharmaceuticals Inc.
Priority to JP5510964A priority Critical patent/JPH07501820A/ja
Priority to EP93900822A priority patent/EP0661988A1/fr
Publication of WO1993011783A1 publication Critical patent/WO1993011783A1/fr

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Classifications

    • 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
    • A61K38/04—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
    • 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]

Definitions

  • This invention relates to biologically active peptides and proteins, and more particularly to compositions and uses
  • composition comprising at least one biologically active amphiphilic peptide or biologically active protein, and a chelating agent.
  • the peptide or protein is preferably an ion channel-forming peptide or protein.
  • a process of inhibiting growth of a target cell in a host which comprises administering to a host at least one biologically active amphiphilic peptide or biologically active protein and a chelating agent.
  • the peptide or protein is
  • the biologically active amphiphilic peptide or biologically active protein and the chelating agent are administered in amounts effective to inhibit growth of a target cell in a host.
  • magnesium ions decrease the activity of certain biologically active amphiphilic peptides as antibacterial agents. It is believed that such decrease in activity may be due to competition between the ions, which are positively charged, and the peptides for negative charges on the bacteria. Applicant also has found that when a chelating agent, which binds ions such as calcium and/or magnesium ions, is added to the biologically active amphiphilic peptide, the activity of the biologically active peptide is enhanced, or potentiated, whereas when known calcium channel-blocking agents, such as verapamil and diltiazem, are added to such peptides, no improvement in the biological activity of the peptides was obtained.
  • a chelating agent which binds ions such as calcium and/or magnesium ions
  • potentiate means either that the biologically active amphiphilic peptide or protein is effective in increasing the biological activity of the chelating agents against a target cell so thereby the chelating agent may be employed in an amount lower than 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 which would be required for preventing, destroying, or inhibiting growth of a target cell.
  • Chelating agents which may be employed in accordance with the present invention are agents which are capable of chelating calcium. Such chelating agents may also increase the
  • Such chelating agents include ethylene dinitrilo tetraacetic acid (EDTA), and ethylene glycol bis- ⁇ -aminoethyl ether N,N,N',N'-tetraacetic acid (EGTA).
  • EDTA ethylene dinitrilo tetraacetic acid
  • EGTA ethylene glycol bis- ⁇ -aminoethyl ether N,N,N',N'-tetraacetic acid
  • 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 structure, sometimes referred to as an ⁇ -helical structure.
  • such peptides have at least 10 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
  • the administration of the biologically active amphiphilic peptides or proteins and chelating agent to a target cell may be by direct administration to the target 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.
  • compositions of the present invention may be any compositions of the present invention.
  • compositions may be used as antimicrobial agents, or in particular as anti-bacterial agents, or as
  • antimicrobial means that the compositions of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as, for example, bacteria.
  • compositions employed in the present invention produce effects adverse to the normal biological functions of the target non-host cell, including death or destruction and prevention of the growth or proliferation of the non-host target cell when contacted with the compositions.
  • compositions of the present invention have a broad range of potent antibiotic activity against a plurality of
  • microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative
  • compositions of the present invention allow a method for treating or controlling microbial infection caused by organisms which are sensitive to the peptides herein described.
  • Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an antimicrobial amount of at least one of the peptides and a chelating agent.
  • compositions may also be used as preservatives or sterilants of materials susceptible to microbial contamination.
  • compositions may be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.
  • a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.
  • compositions may be used topically or systemically and may be in any suitable form such as a liquid, solid,
  • compositions may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful
  • microorganisms including bacteria.
  • compositions of the present invention may be any compositions of the present invention.
  • a host in particular an animal, in an effective antibiotic and/or anti-microbial amount.
  • composition in accordance with the invention will contain an effective anti-microbial amount of one or more of the hereinabove described peptides which have such activity.
  • compositions of the present invention may be used in the treatment of external burns and to treat and/or prevent skin and burn infections.
  • the compositions may be used to treat skin and burn infections caused by organisms such as, but not limited to, P. aeruginosa, S. aureus, and other Streptococcus species.
  • compositions are also useful in the prevention or treatment of eye infections.
  • infections may be caused by bacteria such as, but not limited to, P. aeruginosa, S. aureus, and N. gonorrhoeae.
  • compositions may also be administered to plants in an effective antimicrobial amount to prevent or treat microbial contamination thereof.
  • 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 .05% to 10%.
  • the chelating agent such as those hereinabove described, or derivatives or analogues thereof, when used topically, is
  • the chelating agent is generally employed in a concentration of about .001% to about 25%.
  • the chelating agent is generally employed in an amount of from 1 mg/kg to about 2 grams/kg of host weight per day.
  • the peptide used in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a basic
  • polypeptide having at least ten amino acids wherein the polypeptide includes both hydrophobic and hydrophilic amino acids.
  • the polypeptide may have at least sixteen amino acids wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight
  • 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, 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.
  • the polypeptide chain may comprise 5 or 6 groups of this sequence. In each group, each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.
  • the polypeptide chain preferably has at least 20 amino acids, and no greater than 50 amino acids. It is to be
  • 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.
  • amino acids between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the amino acids does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted group of four amino acids are not spaced from each other.
  • the peptide may have amino acids extending from either end of the chain.
  • the chains may have a Ser-Lys sequence before the "Ala” end, and/or an Ala-Phe sequence after the "Lys" end.
  • Other amino acid sequences may also be attached to the "Ala” and/or the "Lys" end.
  • the chain may have, for example, a C-D sequence before the first A-B-C-D group.
  • other amino acid sequences may be attached to the "A" and/or the "D" end of one of these polypeptide chains.
  • amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other.
  • the peptides may be produced by known techniques and
  • the peptides m y be Synthesized on an automatic synthesizer. Journal of the American Chemical Society, Vol. 85 Pages 2149-54(1963). It is also possible to produce such peptides by genetic engineering techniques.
  • the peptide employed in conjunction with a chelating agent 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 X 12 - - R 11 -R 11 -R 12 -R 13 -R 11 -R 14 -R 12 -R 11 -R 14 - R 12 -R 11 -R 11 -R 14a -(R 15)n -R 14a -R 14 - - wherein 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:
  • R 11 , R 12 , R 14 , and R 14a are as previously defined.
  • a magainin peptide may also have the following structure:
  • X 12 is as previously defined and Z 12 is: (i) 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:
  • 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
  • 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 are described in Proc. Natl. Acad Sci. Vol. 84 pp. 5449-53 (Aug. 1987).
  • magainin peptides refers to the basic magainin structure as well as derivatives and analogs thereof, including but not limited to the representative derivatives or analogs.
  • a chelating agent employed in conjunction with a chelating agent 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 :
  • R 11 , R 12 , R 14 , and R 17 are as previously defined.
  • the PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl end.
  • a PGLa peptide may have the following structure:
  • Y 14 is (i) R 11 ;
  • 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:
  • An XPF peptide is either XPF or an analogue, or derivative thereof.
  • the XPF peptides preferably include the following basic peptide structure X 16 :
  • R 11 , R 12 , R 14 , R 15 and R 17 are as previously defined and R 18 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.
  • an XPF peptide may include the following structure:
  • An XPF peptide may include the following structure:
  • An XPF peptide may also have the following structure:
  • X 16 , Y 16 , and Z 16 are as previously defined: a is 0 or
  • XPF or PGLa peptides which are characterized by the following as listed in the accompanying sequence listing:
  • the peptide employed in conjunction with a chelating agent such as those hereinabove described may be a CPF peptide or appropriate analogue or derviative thereof.
  • 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
  • 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, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids may be Asn, Gln, Ser, and Thr.
  • the basic hydrophilic amino acids may be Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and
  • 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.
  • Y 20 -X 20 - wherein X 20 is the hereinabove described basic peptide structure and Y 20 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 6 additional amino acids at the carboxyl end, which may be represented as follows:
  • X 20 is the hereinabove defined basic peptide structure and Z 20 is
  • 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:
  • X 20 , Y 20 and Z 20 are as Previously defined and a is 0 or 1 and b is 0 or 1.
  • CPF peptides which may be employed in the present invention are represented by the following:
  • 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 31 -R 32 -R 32 ]- n ;
  • X 32 is -[R 32 -R 32 -R 33 -R 31 -R 32 -R 32 -R 31 ]- 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 31 -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), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
  • the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie. Leu, Met, Val, Trp and Tyr,norleucine (Nle), norvaline (Nva), 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:
  • X 31 -Z 31 wherein X 31 is as hereinabove described, and Z 31 is: (i) R 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:
  • 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:
  • 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 :
  • the peptide may include the following structure:
  • n 3 + preferably the peptide has one of the following structures:
  • the biologically active amphiphilic peptide includes the following basic structure X 40 :
  • the peptide may include 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: (i ) R 31 ;
  • the peptide has the following structural formula as indicated in the sequence listing contained herein:
  • the peptide has the following structural formula as indicated in the sequence listing contained herein:
  • the peptide has one of the following structural formulae as indicated in the sequence listing contained herein:
  • n is from 2 to 5.
  • n is 3, and the peptide has the following structural formula:
  • the peptide is selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
  • the peptide includes the following basic structure X 50 :
  • 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 such peptides include those having the following structures:
  • the peptide includes the following basic structure X 52 :
  • R 41 is a hydrophobic amino acid
  • R 42 is a basic hydrophilic or neutral hydrophilic amino acid
  • 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 (i) R 42 ;
  • the peptide may have the following structure:
  • the peptide includes the basic structure X 52 -Z 52 , wherein X 52 is as hereinabove described, and
  • the peptide may have the following structure:
  • the peptide may include the
  • a is 0 or 1
  • b is 0 or 1.
  • the above 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.
  • the peptide employed in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a cecropin.
  • 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.
  • a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a sarcotoxin.
  • the sarcotoxins and analogues and derivatives thereof are
  • 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), a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein, lactoferrin, B- 2 -binding protein, eosinophil cationic protein (ECP), and eosinophil-derived
  • defensins also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein, lactoferrin, B- 2 -binding protein, eos
  • 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.
  • BPI proteins are described in Ooi, et al., J. Biol. Chem., Vol. 262, pgs. 14891-14894 (1981). Perform is described in Henkart, et al. J. Exp. Med., 160:75 (1984), and in Podack, et al. J. Exp. Med., 160:695 (1984). Lactoferrin,
  • B 12 -binding protein eosinophil cationic protein
  • eosinphil-derived neurotoxin are described in Elsbach, et al., Inflammation: Basic Principles and Clinical Correlates, Gallin, et al., eds.; pgs. 445-471 (1988). The above articles are hereby incorporated by reference.
  • ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as
  • the method comprises administering to a host (human or animal) that is being treated with a biologically active peptide or protein an ion selected from the group consisting of calcium ions and magnesium ions.
  • the ion is administered in an amount effective to
  • Such a method is particularly applicable to a method of selective treatment.
  • the peptide or protein may be biologically effective in one or more body parts or organs of a host, but not in others.
  • the ion would be administered to those body parts or organs in which onedid not desire the peptide or protein to- have biological effect.
  • the ion may be administered in an amount up to about 2 grams/kg of host weight.
  • Peptide (1) is (SEQ ID NO: 27)
  • Peptide (2) is (SEQ ID NO: 97) amide-terminated
  • Peptide (3) is (LysIle Ala Gly LysIle Ala) 3 ,
  • each amino acid residue of Peptide (3) is a D-amino acid residue or glycine, were prepared at a concentration of 512 ⁇ g/ml in sterile deionized distilled water and stored at -70°C.
  • the stock peptide solutions are diluted in serial dilutions (1:2) down the wells of a microtiter plate so that the final concentrations of peptides in the wells are 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128, and 256 ⁇ g/ml. 1-5 x 10 5 CFUs/ml of P.
  • aeruginosa ATCC 27853 were added to the wells in full strength or half-strength Mueller Hinton broth (MHB), or in half-strength MHB broth to which 0.002 M Ca 2+ , or 0.002 M Mg 2+ , or 0.001 M Ca 2+ and 0.001M Mg 2+ has been added.
  • the organisms are from a mid-log culture.
  • the inoculum is standardized spectrophotometrically at
  • concentration is defined as the lowest concentration of peptide which produces a clear well in the microtiter plate, The MIC values are given in Table I below.
  • Example 2 The procedure of Example 1 was repeated, except that the assays were carried out in full strength Mueller Hinton Broth (MHB) or cation-adjusted Mueller Hinton Broth (CAMHB). Peptides (1) and (2) were tested for MIC against P. aeruginosa 27853 in each of these broths without further additives, and in each of these broths to which was added 0.05mM, 0.5mM, or 5mM of the calcium channel blockers verapamil or diltiazem. The results are given in Table II below.
  • Peptides (1), (2), (4), (5), and (6) were tested for activity against P. aeruginosa strain 27853 in full or
  • Peptide (4) is amide-terminated Magainin II (SEQ ID NO: 7).
  • Peptide (5) has the following structural formula:
  • Peptide (6) is SEQ ID NO:21, amide-terminated.
  • the results of this assay are given in Table III below:
  • the checkerboard assay was carried out in a 96-well
  • microtiter plate having 12 rows and 8 columns of wells. 100 ⁇ l of plain broth is added to every row of wells. 100 ⁇ l of the desired peptide at 512 ⁇ g/ml is added to the top well, and serially diluted through row 11. 50 ⁇ l of the EDTA in varying
  • Peptides (1) and (2) were tested alone or in combination with EDTA for activity against P. aeruginosa strain 27853.
  • the MIC of Peptide (1) was 64 ⁇ g/ml, and the MIC of EDTA was 4mM.
  • FIC Fractional Inhibitory Index
  • FIC MIC of peptide in combination MIC of EDTA in combination _
  • An FIC value of 0.5 or less is indicative of synergy, a value of greater than 0.5 but less than 2 is indicative of indifference, and a value greater than 2 is indicative of antagonism.
  • Peptide (1) and EDTA were found to be inhibitory, and the FIC values of each combination are given herewith.
  • the assay was also performed to determine the MIC values of Peptide (2), EDTA, and inhibitory combinations thereof.
  • the MIC of Peptide (2) against P. aeruginosa was 4 ⁇ g/ml, and the MIC of EDTA was 8mM.
  • the following combinations of Peptide (2) and EDTA were also found to be inhibitory, and FIC values are given therefor:
  • aeruginosa strain 27853 as employed alone or in combination with 3mM, or 4mM or 5mM EGTA.
  • the procedure followed was that of Example 1, and all testing was done using P. aeruginosa grown in full strength MHB broth. The results of this assay are given in Table IV below.
  • Example 5 The procedure of Example 5 was repeated, except the MIC values of Peptides (1) and (2) were determined for Peptides (1) and (2) alone and in combination with 0.0625mM, 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, or 3mM EGTA. The results of this assay are given in Table V below.
  • ADDRESSEE Carella, Byrne, Bain, Gilfillan,
  • NAME/KEY Magainin II peptide.
  • NAME/KEY magainin peptide
  • NAME/KEY magainin peptide
  • NAME/KEY magainin peptide

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Molecular Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne des combinaisons de peptides ou de protéines amphiphiles biologiquement actifs et d'agents de chélation qui sont utiles comme agents antimicrobiens.
PCT/US1992/010427 1991-12-09 1992-12-03 Composition de peptides biologiquement actifs et d'agents de chelation et procede de traitement associe WO1993011783A1 (fr)

Priority Applications (2)

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JP5510964A JPH07501820A (ja) 1991-12-09 1992-12-03 生物活性ペプチドおよびキレート剤による組成物および治療法
EP93900822A EP0661988A1 (fr) 1991-12-09 1992-12-03 Composition de peptides biologiquement actifs et d'agents de chelation et procede de traitement associe

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US80362991A 1991-12-09 1991-12-09
US803,629 1991-12-09

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5593866A (en) * 1992-08-21 1997-01-14 The University Of British Columbia Cationic peptides and method for production
US5688489A (en) * 1995-09-15 1997-11-18 Resolution Pharmaceuticals, Inc. Non-receptor mediated imaging agents
WO1998020028A3 (fr) * 1996-11-06 1998-07-16 Univ California Clavanines
US6010876A (en) * 1996-11-06 2000-01-04 The Regents Of The University Of California Clavanins
US6191254B1 (en) 1995-08-23 2001-02-20 University Of British Columbia Antimicrobial cationic peptides
US6297215B1 (en) 1995-06-02 2001-10-02 The University Of British Columbia Antimicrobial cationic peptides
WO2005074968A3 (fr) * 2004-02-10 2005-12-08 Univ Maastricht Utilisation medicale de peptides basiques
WO2006008525A1 (fr) * 2004-07-19 2006-01-26 Aq+ Plc Traitement de brulures
WO2012049217A1 (fr) * 2010-10-12 2012-04-19 Consumo Em Verde - Biotecnologia Das Plantas, S.A. Utilisation d'un agent chélateur et de composés antimicrobiens peptidiques
US8318899B2 (en) 2008-01-24 2012-11-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Lytic domain fusion constructs and methods of making and using same
US20160052978A1 (en) * 2010-10-12 2016-02-25 Alexandra CARREIRA Antimicrobial protein
US9492563B2 (en) 2012-10-30 2016-11-15 Esperance Pharmaceuticals, Inc. Antibody/drug conjugates and methods of use

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4810777A (en) * 1987-03-04 1989-03-07 The United States Of America As Represented By The Department Of Health And Human Services Antimicrobial compounds
US5073542A (en) * 1989-06-07 1991-12-17 Magainin Sciences Inc. CPF peptide compositions and their use in inhibiting growth of target cells or a virus
US5114921A (en) * 1988-05-27 1992-05-19 The Children's Hospital Of Philadelphia Amphiphilic peptides and use thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3334879B2 (ja) * 1991-04-15 2002-10-15 アンビー インコーポレイテッド 抗胃障害製剤組成物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4810777A (en) * 1987-03-04 1989-03-07 The United States Of America As Represented By The Department Of Health And Human Services Antimicrobial compounds
US5114921A (en) * 1988-05-27 1992-05-19 The Children's Hospital Of Philadelphia Amphiphilic peptides and use thereof
US5073542A (en) * 1989-06-07 1991-12-17 Magainin Sciences Inc. CPF peptide compositions and their use in inhibiting growth of target cells or a virus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0661988A4 *

Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5593866A (en) * 1992-08-21 1997-01-14 The University Of British Columbia Cationic peptides and method for production
US6906035B2 (en) 1995-06-02 2005-06-14 The University Of British Columbia Antimicrobial cationic peptides
US6465429B1 (en) 1995-06-02 2002-10-15 The University Of British Columbia Antimicrobial cationic peptides
US6297215B1 (en) 1995-06-02 2001-10-02 The University Of British Columbia Antimicrobial cationic peptides
US6191254B1 (en) 1995-08-23 2001-02-20 University Of British Columbia Antimicrobial cationic peptides
US7390873B2 (en) 1995-08-23 2008-06-24 University Of British Columbia Antimicrobial cationic peptides
US5837218A (en) * 1995-09-15 1998-11-17 Resolution Pharmaceuticals Inc. Non-receptor cell mediated imaging agents
US5688489A (en) * 1995-09-15 1997-11-18 Resolution Pharmaceuticals, Inc. Non-receptor mediated imaging agents
US6040293A (en) * 1996-11-06 2000-03-21 The Regents Of The University Of California Clavanins
US6010876A (en) * 1996-11-06 2000-01-04 The Regents Of The University Of California Clavanins
WO1998020028A3 (fr) * 1996-11-06 1998-07-16 Univ California Clavanines
WO2005074968A3 (fr) * 2004-02-10 2005-12-08 Univ Maastricht Utilisation medicale de peptides basiques
WO2006008525A1 (fr) * 2004-07-19 2006-01-26 Aq+ Plc Traitement de brulures
US8318899B2 (en) 2008-01-24 2012-11-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Lytic domain fusion constructs and methods of making and using same
US9255134B2 (en) 2008-01-24 2016-02-09 Esperance Pharmaceuticals, Inc. Lytic domain fusion constructs and methods of making and using same
US8546535B2 (en) 2008-01-24 2013-10-01 Esperance Pharmaceuticals, Inc. Lytic domain fusion constructs and methods of making and using same
EA027441B1 (ru) * 2010-10-12 2017-07-31 Консуму Эм Верди - Биотекноложия Даш Планташ, С.А. Применение хелатирующего агента и антимикробных пептидных соединений
CN110227145A (zh) * 2010-10-12 2019-09-13 肯苏墨艾姆维德-生物技术达思植物有限公司 抗菌蛋白
CN103347387B (zh) * 2010-10-12 2015-04-22 肯苏墨艾姆维德-生物技术达思植物有限公司 螯合剂和肽类抗菌剂化合物的应用
CN103347387A (zh) * 2010-10-12 2013-10-09 肯苏墨艾姆维德-生物技术达思植物有限公司 螯合剂和肽类抗菌剂化合物的应用
US20160052978A1 (en) * 2010-10-12 2016-02-25 Alexandra CARREIRA Antimicrobial protein
US20220000970A1 (en) * 2010-10-12 2022-01-06 Consumo Em Verde Biotecnologia Das Plantas, S.A. Use of chelating agent and peptide antimicrobial compounds
WO2012049217A1 (fr) * 2010-10-12 2012-04-19 Consumo Em Verde - Biotecnologia Das Plantas, S.A. Utilisation d'un agent chélateur et de composés antimicrobiens peptidiques
US11161885B2 (en) * 2010-10-12 2021-11-02 Consumo Em Verde—Biotecnologia Das Plantas, S.A. Antimicrobial protein
US10265376B2 (en) * 2010-10-12 2019-04-23 Consumo Em Verde Biotecnologia Das Plantas, S.A. Use of chelating agent and peptide antimicrobial compounds
US20190263873A1 (en) * 2010-10-12 2019-08-29 Consumo Em Verde - Biotecnologia Das Plantas, S.A. Antimicrobial protein
US20130288954A1 (en) * 2010-10-12 2013-10-31 Consumo Em Vere-Biotecnologia Das Plantas S.A. (Pt/Pt) Use of chelating agent and peptide antimicrobial compounds
US10421792B2 (en) 2010-10-12 2019-09-24 Consumo Em Verde Bio Technologia Das Plantas, S.A. Antimicrobial protein
US11154588B2 (en) * 2010-10-12 2021-10-26 Consumo Em Verde—Biotechnologia Das Plantas, S.A Use of chelating agent and peptide antimicrobial compounds
US10233214B2 (en) 2012-10-30 2019-03-19 Esperance Pharmaceuticals, Inc. Antibody/drug conjugates and methods of use
US9492563B2 (en) 2012-10-30 2016-11-15 Esperance Pharmaceuticals, Inc. Antibody/drug conjugates and methods of use

Also Published As

Publication number Publication date
AU3236293A (en) 1993-07-19
JPH07501820A (ja) 1995-02-23
EP0661988A1 (fr) 1995-07-12
EP0661988A4 (fr) 1995-02-10
CA2125494A1 (fr) 1993-06-24

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