EP0661988A4 - 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.

Info

Publication number
EP0661988A4
EP0661988A4 EP19930900822 EP93900822A EP0661988A4 EP 0661988 A4 EP0661988 A4 EP 0661988A4 EP 19930900822 EP19930900822 EP 19930900822 EP 93900822 A EP93900822 A EP 93900822A EP 0661988 A4 EP0661988 A4 EP 0661988A4
Authority
EP
European Patent Office
Prior art keywords
peptide
ala
lys
amino acids
basic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19930900822
Other languages
German (de)
English (en)
Other versions
EP0661988A1 (fr
Inventor
Barry Berkowitz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Magainin Pharmaceuticals Inc
Original Assignee
Magainin Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Magainin Pharmaceuticals Inc filed Critical Magainin Pharmaceuticals Inc
Publication of EP0661988A4 publication Critical patent/EP0661988A4/fr
Publication of EP0661988A1 publication Critical patent/EP0661988A1/fr
Withdrawn legal-status Critical Current

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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 involving biologically active peptides and chelating agents.
  • 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 preferably an ion channel-forming peptide or protein.
  • 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.
  • 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 permeability of the peptide or protein through the membrane of a target cell, such as a bacterium, as described in Marvin, et al., "Enhanced Penetration of Externally Added Macromolecules Through the Outer Membrane of Gram-Negative Bacteria, " found in Actor, et al. (eds. ) , Antibiotic Inhibition of Bacterial Cell Surface Assembly and Eunction, (1988), pgs. 431-435.
  • 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 o-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.
  • 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.
  • 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.
  • amphiphilic peptide or protein is a peptide which includes both hydrophobic and hydrophilic peptide regions.
  • 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 administered to a host; for example a human or non-human animal, in an amount effective to inhibit growth of a target cell.
  • a host for example a human or non-human animal
  • the compositions may be used as antimicrobial agents, or in particular as anti-bacterial agents, or as antibiotics.
  • 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 bacteria.
  • the 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 be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.
  • a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.
  • Such pharmaceutical compositions may be used topically or 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 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 administered to 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 generally employed in a concentration of about .001% to about 25%. When used systemically, 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 (positively charged) 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 hydroplilic amino acids.
  • the hydrophobic amino acids are in groups of two adjacent amino acids, and each group of two hydrophobic amino acids is spaced from another group of two hydrophobic amino acids by at least one amino acid other than a hydrophobic amino acid (preferably at least two amino acids) and generally by no greater than four amino acids, and the amino acids between pairs of hydrophobic amino acids may or may not be hydrophilic.
  • the hydrophilic amino acids are generally also in groups of two adjacent amino acids in which at least one of the two amino acids is a basic hydrophilic amino acid, with such groups of two hydrophilic amino acids being spaced from each other by at least one amino acid other than a hydrophilic amino acid (preferably at least two amino 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, Gin, Ser, and Thr.
  • the basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu) , and p-aminophenylalanine.
  • Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different.
  • the polypeptide chain may comprise 5 or 6 groups of this sequence. In each group, each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.
  • the polypeptide chain preferably has at least 20 amino acids, and no greater than 50 amino acids. It is to be understood, however, that the polypeptide does not have to consist entirely of the groups described above.
  • the polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention.
  • the groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary provided that in each group of the at least four groups of amino acids there are two hydrophobic and two hydrophilic amino acids as hereinabove noted.
  • the biologically active polypeptide comprises a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each. group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.
  • each of the at least four groups of amino acids which are in the peptide chain is of the sequence A-B-C-D, B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic amino acids, one of C or D is basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid.
  • the resulting polypeptide chain may have one of the following sequences:
  • X is D; C-D- or B-C-D-, Y., is -A or -A-B or -A-B-C
  • X is A-, D-A- or C-D-A-
  • Y is -B, -B-C or B-C-D
  • X 3 is B-, A-B-, D-A-B-
  • Y 3 is -C, -C-D, -C-D-A
  • X 4 is C-, B-C-, A-B-C-
  • Y 4 is -D, -D-A, -D-A-B a is 0 or 1; b is 0 or 1 and n is at least 4.
  • the peptide chain may include amino acids between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the amino acids does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted 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.
  • 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 obtained in substantially pure form.
  • 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-,
  • R-_ is a hydrophobic amino acid, R-. ? is a basic hydrophilic amino acid
  • R 13 is a hydrophobic, neutral hydrophilic, or basic hydrophilic amino acid
  • R... and R 14a are hydrophobic or basic hydrophilic amino acids
  • R ⁇ is glutamic acid or aspartic acid, or a hydrophobic or basic hydrophilic amino acid
  • n is 0 or 1.
  • R... is a hydrophobic or neutral hydrophilic amino acid
  • R- 5 is glutamic acid or aspartic acid.
  • a magainin peptide may include the following structure: v — v 12 12' where X- 7 is the hereinabove described basic peptide structure and Y 12 is
  • a magainin peptide may also have the following structure:
  • X 17 is as previously defined and Z 12 is: (i) R_ fi where R-. fi is a basic hydrophilic amino acid or asparagine or glutamine; or
  • R.g-R.- where R 17 is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid.
  • R-groin 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.. 3 :
  • the magainin peptide may also include the following structure X- 3 -Z 13 ; wherein X 13 is the hereinabove described basic peptide structure and Z.._ is
  • 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.
  • the peptide 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 _-
  • the PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl end.
  • a PGLa peptide may have the following structure:
  • a PGLa like peptide may also have the following structure:
  • a PGLa peptide may also have the following structure: where X 1,4 ⁇ ;' Y1,4. and Z1,4. are as previously defined, a is 0 or
  • An XPF peptide is either XPF or an analogue, or derivative thereof.
  • the XPF peptides preferably include the following basic peptide structure X..,. :
  • 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: where Xl..o,, Y.l , and Zl.b _. 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.
  • R 21 ⁇ R 2l R 23 "R -2l "R 2l”R 24 ⁇ R 25 "R 21 ⁇ ⁇ wherein R 21 is a hydrophobic amino acid; R 22 is a hydrophobic amino acid or a basic hydrophilic amino acid;
  • R Dust 3 is a basic hydrophilic amino acid
  • R 24 is a hydrophobic or neutral hydrophilic amino acid
  • R 7 _. 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, Gin, Ser, and Thr.
  • the basic hydrophilic amino acids may be Lys, Arg, His, Orn, homoarginine (Har), 2, -diaminobutyric acid (Dbu), and -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:
  • R 22 -R 21 -R 22 -R 25 preferably Glycine - R 21 - 22 - R 25 -
  • R 2 l' R 22' and R 25 are as P reviously defined.
  • the carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids.
  • the basic structure may have from 1 to 6 additional amino acids at the carboxyl end, which may be represented as ollows:
  • X 20 is the hereinabove defined basic peptide structure
  • R 21 -R 21 -R 24 -R 24 -R 26 -Gln-Gln wherein R 2 _ and R 24 are as previously defined, and R 2 _ 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:
  • 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 3 _ wherein: ⁇
  • X 31 is "lR 3l “”R 32 ⁇ R 32 ⁇ R 33 ⁇ R 31 ⁇ R 32 ⁇ R 32 ] ⁇ n ;
  • X 32 is " [ R 32 "R 32 “R 33 “”R 3l “R 32 “R 32 “R 31 ⁇ “ n ;
  • X 33 is “ [ R 32 “R 33 ⁇ R 3l “R 32 “R 32 ⁇ R 3l “R 32 ] " n
  • X 34 is " [ R 33 ⁇ R 3l “R 32 “R 32 “R 3l “”R 32 “R 32 ] ⁇ n
  • 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, Gin, Ser and Thr.
  • the peptide when the peptide includes the structure X,.. , the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 31 the peptide may include the following structure:
  • the peptide may include the following structure:
  • Y 32 " X 3 2 ' w ⁇ erein X 3 2 is as hereinabove described, and Y 32
  • the peptide when the peptide includes the structure X 32 , the peptide may include the following structure: X 32 ⁇ Z 3 2 ' wherein X 32 is a ⁇ hereinabove described, and Z 32 is:
  • the peptide may nclude, the following structure: Y 32 ⁇ a " X 32 " Z 32 b' whe ein Y 3 2 and Z 32 are S P revious ly defined, a is 0 or 1, and b is 0 or 1.
  • 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:
  • the peptide when the peptide includes the structure X 35 , the peptide may include the following
  • the peptide may include the following structure:
  • n X 35 and Z 35 are as previously defined, a is 0 or 1, and b is 0 or 1.
  • the peptide when the peptide includes the structure X J-,O-, the peptide may include the following
  • 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 3 _-X 37 , wherein X 37 is as hereinabove described, and Y 37 is: (i) R 32 ; (ii) R 31 -R 32 ; (iii) R 33 -R 31 - R 32 ; (iv) R 32 - R 33 -R 31 -R 32 ; (v) R 32 _R 32 "R 33 "R 3l "R 32 ; ° r (vi) R 31 -R 32 -R 32 -R 33 -R 31 -R 32 , wherein R 31 , R 32 , and R 33 are as hereinabove described.
  • the peptide when the peptide includes the structure X 37 , the peptide may include the following structure:
  • R 32 - R 31 - 32- R 32 (iv) R 32 - R 31 - 32- R 32 ; (v) R 32 "R 31 ⁇ R 32 "R 32 "R 33' * or (vi) R 32 -R 31 -R 32 -R 32 -R 33 -R 31 .
  • 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 R 31 ⁇ R 32 ⁇ R 32 ⁇ R 33 "R "4 ⁇ R 32 ⁇ R 32 ⁇ R 31 ⁇ R 32 ⁇ R 32 ⁇ R 32 _R 34 ⁇ R 32 ⁇ R 32' wherein R 31 R 2 ' a ( - R 3 are as hereinabove described, and R_ 4 is a basic hydrophilic or hydrophobic amino acid.
  • the peptide may include the following structure:
  • the peptide may include the following structure:
  • X 4 _-Z 40 wherein X 40 is as hereinabove described and Z 4Q is: (i ) R 31 ; ( ii ) R 31 -R 32 ; (iii ) R 31 -R 32 -R 32 ; ( iv) R 31 -R 32 -R 32 - R 33 ;
  • peptide has the following structural formula as indicated in the sequence listing contained herein: (SEQ ID NO:69)-NH 2 .
  • 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 :
  • the peptide includes the basic structure Y 50 ⁇ X 50' wherein X 5Q is as hereinabove described and Y 5Q 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 ⁇ 2 :
  • R 42 R 41 ⁇ R 42 "R 42 “R 41 ⁇ R 4l “R 42 “R 42 “R 4l “R 42 “R 42' wherein R 41 is a hydrophobic amino acid, and R 42 is a basic hydrophilic or neutral hydrophilic amino acid.
  • R... is leucine.
  • R 42 is lysine
  • the peptide includes the basic structure
  • Y 5 2 ⁇ X 5 52 2 '' W w h h € erein X lake is as hereinabove described, and Y 52 is (ii) R 41 -R 42 ; or
  • 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 ⁇ 52 ⁇ Z 52' wherein X 52 is as hereinabove described, and
  • the peptide may have the following structure: Lys Leu Lys Lys Leu Leu Lys Lys Leu
  • the peptide may include the structure:
  • the above peptides may be acetylated with a CH_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.
  • the peptide employed in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a sarcotoxin.
  • a chelating agent such as those hereinabove described, or derivatives or analogues thereof.
  • the sarcotoxins and analogues and derivatives thereof are described in Molecular Entomology, pages 369-78, in particular p. 375 Alan R. Lis ⁇ 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), a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein, lactoferrin, B- 2 -binding protein, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin.
  • HNP human neutrophil antimicrobial peptides
  • MBP major basic protein
  • BPI bactericidal permeability-increasing protein
  • ECP eosinophil cationic protein
  • Defensins are described in Selsted, et al., J. Clin. Invest. , Vol. 76, pgs.
  • 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).
  • Perform is described in Henkart, et al. J. Ex . Med. , 160:75 (1984), and in Podack, et al. J. Exp. Med. , 160:695 (1984).
  • Lactoferrin, B 12 -binding protein, eosinophil cationic protein, and 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 analogues or derivatives.
  • a method of antagonizing the biological activity of a biologically active amphiphilic peptide or protein, such as those hereinabove described, in a host 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 antagonize the biological activity of the peptide or protein.
  • Such a method is particularly applicable to a method of selective treatment. For example, one may wish for the peptide or protein to 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 one did not desire the peptide or protein to- have biological effect.
  • the ion would be administered to those body parts or organs in which one did 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 (Lys He Ala Gly Lys He Ala) 3 , wherein 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
  • MIC MIC for each peptide is determined.
  • Minimal inhibitory concentration is defined as the lowest concentration of peptide which produces a clear well in the microtiter plate, The 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. Table II
  • Example 3 Peptides (1), (2), (4), (5), and (6) were tested for activity against P. aeruginosa strain 27853 in full or half-strength Mueller Hinton Broth according to the procedure of Example 1, and in Mueller Hinton Broth to which 0.034mM, or 0.34mM, or 3. mM of ethylene dinitrilo tetraacetic acid (EDTA) or ethylene glycol bis (B-aminoethyl ether) NNN'N'-tetraacetic acid is added.
  • Peptide (4) is amide-terminated Magainin II (SEQ ID NO:7).
  • Peptide (5) has the following structural formula: Gly He Gly D-Lys D-Phe Leu His Ser
  • Peptide (6) is SEQ ID N0:21, 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. lOO ⁇ l of plain broth is added to every row of wells. lOO ⁇ 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 concentrations (obtained through serial dilutions), are added to each appropriate column of wells. 50 ⁇ l of P. aeruginosa strain 27853 is then added to each well. The plate is then 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.
  • 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
  • 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.
  • Example 5 Peptides (1) and (2) were tested for MIC against P. 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, ImM, 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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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.
EP93900822A 1991-12-09 1992-12-03 Composition de peptides biologiquement actifs et d'agents de chelation et procede de traitement associe Withdrawn EP0661988A1 (fr)

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US80362991A 1991-12-09 1991-12-09
US803629 1991-12-09
PCT/US1992/010427 WO1993011783A1 (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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US5593866A (en) * 1992-08-21 1997-01-14 The University Of British Columbia Cationic peptides and method for production
US6057291A (en) 1995-06-02 2000-05-02 University Of British Columbia Antimicrobial cationic peptides
CA2230160A1 (fr) 1995-08-23 1997-03-06 The University Of British Columbia Peptides cationiques antimicrobiens et procedes de reconnaissance de ceux-ci
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
WO2005074968A2 (fr) * 2004-02-10 2005-08-18 Universiteit Maastricht Utilisation medicale de peptides basiques
GB0415985D0 (en) * 2004-07-19 2004-08-18 Taylor Russell Methods of manufacture and applications of a chelating compounds or substances that when used as a treatment for burns will assist in the growth
PT2252627T (pt) 2008-01-24 2017-07-24 Esperance Pharmaceuticals Constructos de fusão de domínio lítico e métodos para produzir e utilizar os mesmos
WO2012049215A1 (fr) * 2010-10-12 2012-04-19 Consumo Em Verde - Biotecnologia Das Plantas, S.A. Protéine antimicrobienne
PT105332A (pt) * 2010-10-12 2012-04-12 Cev Biotecnologia Das Plantas S A Agentes para utilização com antimicrobianos
KR102460297B1 (ko) 2012-10-30 2022-10-28 에스퍼란스 파마슈티컬스, 인코포레이티드 항체/약물 컨쥬게이트 및 이의 사용 방법

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WO1992018143A1 (fr) * 1991-04-15 1992-10-29 Applied Microbiology, Inc. Compositions pharmaceutiques contre les troubles gastriques

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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

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018143A1 (fr) * 1991-04-15 1992-10-29 Applied Microbiology, Inc. Compositions pharmaceutiques contre les troubles gastriques

Non-Patent Citations (2)

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Title
FINK J ET AL: "The chemical synthesis of cecropin D and an analog with enhanced antibacterial activity.", J BIOL CHEM (UNITED STATES), APR 15 1989, VOL. 264, NO. 11, PAGE(S) 6260-7, *
See also references of WO9311783A1 *

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WO1993011783A1 (fr) 1993-06-24

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