WO1992000090A1 - Composition and treatment with biologically active peptides and antibiotics which inhibit dna gyrase - Google Patents
Composition and treatment with biologically active peptides and antibiotics which inhibit dna gyrase Download PDFInfo
- Publication number
- WO1992000090A1 WO1992000090A1 PCT/US1991/004453 US9104453W WO9200090A1 WO 1992000090 A1 WO1992000090 A1 WO 1992000090A1 US 9104453 W US9104453 W US 9104453W WO 9200090 A1 WO9200090 A1 WO 9200090A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- antibiotic
- amino acid
- dna gyrase
- composition
- Prior art date
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 156
- 239000000203 mixture Substances 0.000 title claims abstract description 32
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 26
- 102000004196 processed proteins & peptides Human genes 0.000 title description 37
- 229940088710 antibiotic agent Drugs 0.000 title description 10
- 108010054814 DNA Gyrase Proteins 0.000 claims abstract description 52
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 49
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 49
- 230000003115 biocidal effect Effects 0.000 claims abstract description 48
- 150000001413 amino acids Chemical class 0.000 claims description 135
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims description 54
- 230000002209 hydrophobic effect Effects 0.000 claims description 37
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- 108060003100 Magainin Proteins 0.000 claims description 20
- 230000007935 neutral effect Effects 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 16
- KYGZCKSPAKDVKC-UHFFFAOYSA-N Oxolinic acid Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC2=C1OCO2 KYGZCKSPAKDVKC-UHFFFAOYSA-N 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 10
- 239000003306 quinoline derived antiinfective agent Substances 0.000 claims description 7
- 101500009721 Xenopus laevis Magainin-2 Proteins 0.000 claims description 4
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- 229960000210 nalidixic acid Drugs 0.000 claims description 4
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical group C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 claims description 4
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- 229960004621 cinoxacin Drugs 0.000 claims description 3
- VDUWPHTZYNWKRN-UHFFFAOYSA-N cinoxacin Chemical group C1=C2N(CC)N=C(C(O)=O)C(=O)C2=CC2=C1OCO2 VDUWPHTZYNWKRN-UHFFFAOYSA-N 0.000 claims description 3
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 9
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- 230000002401 inhibitory effect Effects 0.000 description 6
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- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000001974 tryptic soy broth Substances 0.000 description 4
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- OFIZOVDANLLTQD-ZVNXOKPXSA-N magainin i Chemical compound C([C@H](NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O)C1=CC=CC=C1 OFIZOVDANLLTQD-ZVNXOKPXSA-N 0.000 description 3
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- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
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- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
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- QKDHBVNJCZBTMR-LLVKDONJSA-N (R)-temafloxacin Chemical compound C1CN[C@H](C)CN1C(C(=C1)F)=CC2=C1C(=O)C(C(O)=O)=CN2C1=CC=C(F)C=C1F QKDHBVNJCZBTMR-LLVKDONJSA-N 0.000 description 1
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- WUWFMDMBOJLQIV-UHFFFAOYSA-N 7-(3-aminopyrrolidin-1-yl)-1-(2,4-difluorophenyl)-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid Chemical compound C1C(N)CCN1C(C(=C1)F)=NC2=C1C(=O)C(C(O)=O)=CN2C1=CC=C(F)C=C1F WUWFMDMBOJLQIV-UHFFFAOYSA-N 0.000 description 1
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 description 1
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- VUFOSBDICLTFMS-UHFFFAOYSA-M ethyl-hexadecyl-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)CC VUFOSBDICLTFMS-UHFFFAOYSA-M 0.000 description 1
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- ZEKZLJVOYLTDKK-UHFFFAOYSA-N lomefloxacin Chemical compound FC1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNC(C)C1 ZEKZLJVOYLTDKK-UHFFFAOYSA-N 0.000 description 1
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- FHFYDNQZQSQIAI-UHFFFAOYSA-N pefloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCN(C)CC1 FHFYDNQZQSQIAI-UHFFFAOYSA-N 0.000 description 1
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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
- A61K38/04—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
- 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 involving biologically active peptides or proteins and an antibiotic which inhibits DNA gyrasee , and in particular quinolone antibiotics such as ciprofloxacin.
- composition which includes includes at least one biologically active amphiphilic peptide and/or biologically active protein ; and an antibiotic which inhibits DNA gyrase.
- a process wherein there is administered to a host at least one biologically active amphiphilic peptide which is an ion channel forming peptide and/or biologically active protein; and an antibiotic which inhibits DNA gyrase.
- An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the permeability for ions across a natural or synthetic lipid membrane.
- B Christensen et al. PNAS Vol. 85 Pgs 5072-76 (July, 1988) describes methodology which indicates whether or not a peptide or protein has ion channel-forming properties and is therefore an ionophore.
- an ion channel- forming peptide or ion channel forming protein is a peptide or protein which has ion channel- forming properties as determined by the method of Christensen et al.
- An amphiphilic peptide is a peptide which includes both hydrophobic and hydrophilic peptide regions.
- biologically active peptide or protein, and an antibiotic which inhibits DNA gyrase are administered to a host
- biologically active peptide or protein and the antibiotic which inhibits DNA gyrase may be administered as a single composition or in separate compositions, and the single or separate compositions may include additional materials, actives and/or inactives, in addition to the peptide and/or protein and antibiotic which inhibits DNA gyrase.
- the ion channel- forming peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water.
- such peptides are non-hemolytic; i. e. , they will not rupture blood cells at effective concentrations .
- 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.
- such peptides have at least 16 amino acids, and preferably at least 20 amino acids. In most cases, such peptides do not have in excess of 40 amino acids.
- DNA gyrase is an enzyme which is involved in the formation of bonds between individual coiling strands of replicating bacterial DNA
- DNA gyrase is necessary for the normal replication of bacterial DNA, and, therefore, antibiotics which inhibit DNA gyrase inhibit the normal replication of bacterial DNA.
- antibiotics which inhibit DNA gyrase include nalidixic acid, oxolinic acid, cinoxacin, and quinolone antibiotics which include ciprofloxacin, norfloxacin, ofloxacin, enoxacin, pefloxacin, lomefloxacin, fleroxacin, tosulfloxacin, temafloxacin, and rufloxacin.
- the following structural formulae of representative examples of antibiotics which inhibit DNA gyrase are the following structural formulae of representative examples of antibiotics which inhibit DNA gyrase.
- Nalidixic acid has the following structure:
- Oxolinic acid has the following structure:
- Ciprofloxacin has the following structure:
- Norfloxacin has the following structure:
- the antibiotic which inhibits DNA gyrase is a quinolone antibiotic, and most preferably, the quinolone antibiotic is ciprofloxacin.
- the peptide or protein and the antibiotic which inhibits DNA gyrase are employed in amounts effective to inhibit and/or prevent and/or destroy the growth of a target cell.
- the quinolone antibiotic potentiates the action of the peptide or protein, and the peptide or protein potentiates the action of the antibiotic which inhibits DNA gyrase.
- potentiate means that the amount of antibiotic which inhibits DNA gyrase is effective to reduce the minimum effective concentration of the peptide or protein for inhibiting growth of a target cell and the amount of peptide or protein is effective to reduce the minimum effective concentration of the antibiotic which inhibits DNA gyrase for inhibiting growth of a target cell.
- the peptide or protein is administered topically at a concentration of from .05% to 10%.
- the peptide or protein is employed to provide peptide or protein dosages of from lmg to 500mg per kilogram of host weight.
- the antibiotic which inhibits DNA gyrase in general, is used topically at a concentration of from 0.05% to 10%.
- the antibiotic which inhibits DNA gyrase is generally employed in an amount of from 1.25 to 45mg per kilogram of host weight per day.
- a combination of peptide or protein and antibiotic which inhibits DNA gyrase 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 Gram -negative bacteria. Such compositions may be employed for treating or controlling microbial infection caused by organisms which are sensitive to such compositions.
- 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 antibiotic which inhibits DNA gyrase.
- compositions may also be used as preservatives or sterilants for materials susceptible to microbial contamination .
- compositions of the present invention may also be used in the treatment of external burns and to treat and/or prevent skin and burn infections.
- the compositions may be used to treat skin and burn infections caused by organisms such as, but not limited to, P . aeruginosa and S. aureus.
- compositions may also be used in the prevention or treatment of eye infections.
- infections may be caused by bacteria such as , but not limited to, P. aeruginosa. S .auerus. and N . gonorrhoeae.
- the peptide used in conjunction with the antibiotic which inhibits DNA gyrase 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.
- 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, He, Leu, Met, Val, Trp, and Tyr.
- 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 and ornithine (O) .
- 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 .
- 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 a basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid.
- the resulting polypeptide chain may have one of the following sequences:
- X 1 is D; C-D- or B-C-D- , Y. is -A or -A-B or -A-B-C
- 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-BY 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 sequence before th e "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.
- the peptides may be produced by known techniques and obtained in substantially pure form.
- the peptides may be synthesized on an automatic synthesizer. Journal of 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 an antibiotic which inhibits DNA gyrase 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 preferably 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 hydrophilic, or basic hydrophilic amino acid
- R 14 and R 14a are hydrophobic or basic hydrophilic amino acids
- R 15 is glutamic acid or aspartic acid, or a hydrophobic or a basic hydrophilic amino acid
- n is 0 or 1.
- R 13 is a hydrophobic or neutral hydrophilic amino acid
- R 14a is a hydrophobic 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:
- R 16 where R 16 is a basic hydrophilic amino acid or asparagine or glutamine .
- 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 14 -R 1 1 -R 14a -R 12 -R 1 1 -R 1 1 -R 12 -R 13 -
- R 11 -R 14 -R 12 -R 1 1 -R 1 1 -R 12 - are amino acids as hereinabove described.
- the magainin peptide may also include the following structure
- R 17 (R 17 ) n wherein R 11 , R 14 , R 14a , R 15 , R 16 , and R 1 7 are as hereinabove described, and n is 0 or 1, and each n may be the same or different.
- the magainin peptides generally include at least fourteen amino acids and may include up to forty amino acids.
- a magainin peptide preferably has 22 or 23 amino acids. Accordingly, the hereinabove described basic peptide structures of a magainin peptide may include additional amino acids at the amino end or at the carboxyl end, or at both ends .
- magainin peptides having the following primary sequence (expressed as a single letter code) as well as appropriate analogues and derivatives thereof :
- the peptide employed in conjunction with an antibiotic which inhibits DNA gyrase 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 11 -R 12 - where R 11 , R 12 , R 1 4 , 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 followin g structure:
- a PGLa 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
- R 11 , R 12 , R 14 , R 15 and R 17 are as Prevlously defined and R 18 is glutamine or asparagine or a basic hydrophilic, or hydrophobic amino acid and, n is 0 or 1.
- the XPF peptides generally include at least nineteen amino acids and may include up to forty amino acids . Accordingly, the hereinabove described basic peptide structure of XPF may include additional amino acids at the amino end, or at the carboxyl end or at both the amino and carboxyl ends. Thus, for example, 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:
- XPF or PGLa peptides which are characterized by the following primary amino acid sequence (single letter amino acid code) :
- the peptide employed in conjunction with an antibiotic which inhibits DNA gyrase may be a CPF peptide or appropriate analogue or derviative thereof .
- CPF peptides as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
- the CPF peptide 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.
- hydrophobic amino acids are Ala, Cys, Phe, Gly, Ile, Leu, Met, Val, Trp, and Tyr.
- the neutral hydrophilic amino acids are Asn, Gin, Ser, and Thr.
- the basic hydrophilic amino acids are Lys, Arg, His, and ornithine.
- 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:
- the carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids.
- the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented follows:
- X 30 is the hereinabove defined basic peptide structure and Z 30 is
- R 21 -R 21 -R 24 -R 24 -R 26 -Gln (vi) R 21 -R 21 -R 24 -R 24 -R 26 -Gln; or (vii) R 21 -R 21 -R 24 -R 24 -R 26 -Gln-Gln,
- 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 are useful in the present invention some of which have been described in the literature and comprise the following sequences (single letter amino acid code) :
- CPF peptides which may be employed in the present invention are represented by the following (single letter amino acid code) : G12S3LG4ALKA5LKIG678LGG9(10)QQ
- CPF peptide includes the basic peptide structure as well as analogs or derivatives thereof .
- the biologically active peptide may include the following basic strucutre X 40 :
- R 41 -R 42 -R 42 -R 43 -R 41 -R 42 -R 42 ] n wherein R 41 is a basic hydrophilic amino acid, R 42 is a hydrophobic amino acid, R 43 is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5.
- such peptide may include the following structure :
- Y 40 -X 40 wherein X 40 is as hereinabove described, and Y 40 is:
- such peptide may include the following structure:
- X 40 - Z 40 wherein X 40 is as hereinabove described, and Z 40 is:
- such peptide may include the following structure:
- n 3 or 3 or 4 or 5 or 6 or 7 or 8 or 10 or 11 or 12 or 13 or 14 or 15 or 14 or 15 or 16 or 15 or 16 or 16 or 17 or 18 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or 19 or 20 or
- n 2
- the peptide preferably is of the following structure as indicated by the single letter amino acid code:
- the biologically active amphiphilic peptide may be a biologically active amphiphilic peptide including the following basic structure X 50 : R 41 -R 42 -R 42 -R 43 -R 4l -R 42 -R 42 -R 41 -R 42 -R 42 -R 42 -R 41 -R 42 -R 42 ,
- R 41 , R 42 and R 43 are as hereinabove described.
- such peptide may include the following structure:
- Y 50 -X 50 wherein X 50 is as hereinabove described, and Y 50 is :
- such peptide may include the following structure:
- X 50 -Z 50 wnerein X 50 is as hereinabove described and Z 50 is :
- the peptide may include the following structure: ( Y 50 )a-X 50- (Z 50 )b , wherein X and Y are as previously defined, a is
- the peptide is of the following structural formula as indicated by the single letter amino acid code :
- the peptide is of the following structural formula as indicated by the single letter amino acid code:
- the peptide employed in conjunction with an antibiotic which inhibits DNA gyrase is a cecropin.
- the cecroptns and analogs 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.
- cecropins includes the basic structure as well as analogues and derivatives.
- the peptide employed in conjunction with an antibiotic which inhibits DNA gyrase is a sarcotoxin .
- the sarcotoxins and analogs 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.
- each of the amino acid residues of the biologically active amphiphilic peptide structures hereinabove described is a D- amino acid residue or a glycine residue.
- an ion channel-forming protein may be used in conjunction with an antibiotic which inhibits DNA gyrase .
- Ion channel- forming proteins which may be employed include defensins, also known as human neutrophil antimicrobial peptides (HNP) , major basic protein (MBP) of eosinophils, bactericidal permeability -increasing protein (BPI) , and a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein .
- HNP human neutrophil antimicrobial peptides
- MBP major basic protein
- BPI bactericidal permeability -increasing protein
- a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein .
- Defensins are described in Selsted, et al. , J . Clin. Invest. . Vol. 76, pgs. 1436-1439 (1985) .
- MBP proteins are described in Wasmoen, et al. ,
- BPI proteins are described in Ooi, et al, J. Biol. Chem. . Vol . 262 , pgs. 14891-14894 (1987) .
- Perforin is described in Henkart, et al. J. Exp. Med. , 160: 75 (1984) , and in Podack, et al. , J. Exp . Med. 160:695 (1984) .
- the above articles are hereby incoroporated by reference.
- ion channel-forming proteins includes the basic structures of the ion -forming proteins as well as analogues and derivatives.
- CFU's colony forming units
- P. aeruginosa strain 27853 or of P. aeruginosa strain 107 (which is gentamicin - resistant) dispersed in 100 ul of trypticase soy broth (TSB) were added to each of a series of test wells.
- Either Peptide 1, Peptide 2, or Peptide 3 was added to each test well in increasing amounts from 0.25 to 256 ⁇ g/ml in absence of or in the presence of 20% of the minimal inhibitory concentration (MIC) of ciprofloxacin.
- Peptide 1 is amide-terminated Magainin II
- Peptide 2 is of the following structural formula:
- Peptide 3 is of the following structural formula:
- the MIC of ciprofloxacin alone against P. aeruginosa strain 27853 was l ⁇ g/ml, and against P. aeruginosa strain 107 was 2 ⁇ g/ml.
- the MIC values for Peptides 1 , 2 and 3 either alone or in combination with 20% of the MIC of ciprofloxacin are given in Table I below.
- microorganisms employed in the assays were grown according to the procedure described in Stutman, et al.
- Examples 2 through 4 organisms were subcultured on agar plates, and then grown in trypticase soy broth (TSB) , or Mueller-Hinton broth. The final assays were then conducted in microtiter plates containing Mueller-Hinton broth 10 organisms were added to each well.
- TTB trypticase soy broth
- the minimal inhibitory concentrations (MIC's) of ciprofloxacin alone, of Peptide 1, as hereinabove described in Example 1 , alone, of ciprofloxacin when Peptide 1 was added (ciprofloxacin + Peptide 1) , and of Peptide 1 when ciprofloxacin was added, (Peptide 1 + ciprofloxacin) were tested against various isolates of strain MR- PSA Pseudomonas aeruginosa.
- the second compound (or "+ " compound) dose to establish synergy was the lowest dose of the synerglzing drug which alone lacked antibacterial effect and was at least a 50% lower dose than the MIC for the synerglzing agent alone. The results are given below in Table 2.
- the peptide or protein and antibiotic which inhibits DNA gyrase may be employed for treating a wide variety of hosts.
- a host is an animal, and such animal may be a human or non-human animal.
- the peptide or protein and the antibiotic which inhibits DNA gyrase may be employed together in a single composition, or in separate compositions .
- the antibiotic which inhibits DNA gyrase and the peptide or protein may be delivered or administered in different forms, for example, the antibiotic which inhibits DNA gyrase may be administered systemically, while the peptide or protein may be administered topically.
- the peptide or protein and/or antibiotic which inhibits DNA gyrase 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 which inhibits DNA gyrase 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, in particular bacteria .
- the peptide(s) or protein(s) of the present invention may be administered to a host; in particular an animal, in an effective anti-microbial, in particular in an anti-bacterial amount, in conjunction with an antibiotic which inhibits DNA gyrase, for potentiating the activity of the peptide or protein.
- the peptide could be administered in an amount of up to about 1% weight to weight and the antibiotic which inhibits DNA gyrase delivered in an amount of about 50 mM (about 0.1%) .
- the antibiotic which inhibits DNA gyrase could be administered topically in conjunction with systemic administration of the peptide and/or protein.
- the peptide or protein may be administered IV or IP to achieve a serum dose of 100 micrograms per milliliter (10 milligrams per kilogram) in conjunction with a topical dose of antibiotic which inhibits DNA gyrase of from about 4 ⁇ g/ml to about 100 ⁇ g/ml.
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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 which inhibits DNA gyrase. The biologically active amphiphilic peptide and the antibiotic which inhibits DNA gyrase may be administered in amounts effective to inhibit growth of a target cell such as a bacterium.
Description
COMPOSITION AND TREATMENT WITH BIOLOGICALLY
ACTIVE PEPTIDES AND ANTIBIOTICS WHICH INHIBIT
DNA GYRASE
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 which inhibits DNA gyrasee , and in particular quinolone antibiotics such as ciprofloxacin.
In accordance with an aspect of the present invention, there is provided a composition which includes includes at least one biologically active amphiphilic peptide and/or biologically active protein ; and an antibiotic which inhibits DNA gyrase.
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 amphiphilic peptide which is an ion channel forming peptide and/or biologically active protein; and an antibiotic which inhibits DNA gyrase.
An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the permeability for ions across a natural or synthetic lipid membrane. B . Christensen et al. PNAS Vol. 85 Pgs 5072-76 (July, 1988) describes methodology which indicates whether or not a peptide or protein has ion channel-forming properties and is therefore an ionophore. As used herein an ion channel- forming peptide or ion channel forming protein is a peptide or protein which has ion channel- forming properties as determined by the method of Christensen et al.
An amphiphilic peptide is a peptide which includes both hydrophobic and hydrophilic peptide regions.
In accordance with an aspect of the present invention wherein the biologically active peptide or protein, and an antibiotic which inhibits DNA gyrase are administered to a host, such biologically active peptide or protein and the antibiotic which inhibits DNA gyrase may be administered as a single composition or in separate compositions, and the single or separate compositions may include additional materials, actives and/or inactives, in addition to the peptide and/or protein and antibiotic which inhibits DNA gyrase.
The ion channel- forming peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water. In addition, such peptides are non-hemolytic; i. e. , they will not rupture blood cells at effective concentrations . 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 structure.
In general, such peptides have at least 16 amino acids, and preferably at least 20 amino acids. In most cases, such peptides do not have in excess of 40 amino acids.
DNA gyrase is an enzyme which is involved in the formation of bonds between individual coiling strands of replicating bacterial DNA Thus, DNA gyrase is necessary for the normal replication of bacterial DNA, and, therefore, antibiotics which inhibit DNA gyrase inhibit the normal replication of bacterial DNA.
Examples of antibiotics which inhibit DNA gyrase include nalidixic acid, oxolinic acid, cinoxacin, and quinolone antibiotics which include ciprofloxacin, norfloxacin, ofloxacin, enoxacin, pefloxacin, lomefloxacin, fleroxacin, tosulfloxacin, temafloxacin, and rufloxacin. The following structural formulae of representative examples of antibiotics which inhibit DNA gyrase.
Nalidixic acid has the following structure:
Oxolinic acid has the following structure:
Of the antibiotics which inhibit DNA gyrase which are also quinolone antibiotics, the following are representative structural formulae.
Ciprofloxacin has the following structure:
Norfloxacin has the following structure:
Antibiotics which inhibit DNA gyrase are further described in Clinical and Infectious Diseases. W. B . Saunders Co . (1987) . In a preferred embodiment, the antibiotic which inhibits DNA gyrase is a quinolone antibiotic, and most preferably, the quinolone antibiotic is ciprofloxacin.
In employing both an ion channel-forming biologically active amphiphilic peptide or an ion channel- forming protein , and an antibiotic which inhibits DNA gyrase, whether administered or prepared in a single composition, or in separate compositions, the peptide or protein and the antibiotic which inhibits DNA gyrase are employed in amounts effective to inhibit and/or prevent and/or destroy the growth of a target cell. In effect, the quinolone antibiotic potentiates the action of the peptide or protein, and the peptide or protein potentiates the action of the antibiotic which inhibits DNA gyrase. The term "potentiate, " as employed herein, means that the amount of antibiotic which inhibits DNA gyrase is effective to reduce the minimum effective concentration of the peptide or protein for inhibiting growth of a target cell and the amount of peptide or protein is effective to reduce the minimum effective concentration of the antibiotic which inhibits DNA gyrase for inhibiting growth of a target cell.
In general, the peptide or protein is administered topically at a concentration of from .05% to 10%. When administered systemically, the peptide or protein is employed to provide peptide or protein dosages of from lmg to 500mg per kilogram of host weight.
The antibiotic which inhibits DNA gyrase, in general, is used topically at a concentration of from 0.05% to 10%. When used systemically,
the antibiotic which inhibits DNA gyrase is generally employed in an amount of from 1.25 to 45mg per kilogram of host weight per day.
The use of a combination of peptide or protein and antibiotic which inhibits DNA gyrase, 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 Gram -negative bacteria. Such compositions may be employed for treating or controlling microbial infection caused by organisms which are sensitive to such compositions. 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 antibiotic which inhibits DNA gyrase.
The compositions may also be used as preservatives or sterilants for materials susceptible to microbial contamination .
The compositions of the present invention may also be used in the treatment of external burns and to treat and/or prevent skin and burn infections. In particular, the compositions may be used to treat skin and burn infections caused by organisms such as, but not limited to, P . aeruginosa and S. aureus.
Such compositions may also be used in the prevention or treatment of eye infections. Such infections may be caused by bacteria such as , but not limited to, P. aeruginosa. S .auerus. and N . gonorrhoeae.
In accordance with a preferred embodiment, the peptide used in conjunction with the antibiotic which inhibits DNA gyrase 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, He, Leu, Met, Val, Trp, and Tyr. 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 and ornithine (O) .
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 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.
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 a basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid. The resulting polypeptide chain, therefore, may have one of the following sequences:
(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- , Y. 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
X3 is B- , A-B- , D-A-BY3 is -C , -C-D, -C-D-A
X4 is C- , B-C- , A-B-C- Y4 is -D, -D-A, -D-A-B
a is o or 1; b is o 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
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.
III Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala-
IV Ser-Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala-Phe-
V Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser
The peptide, may have amino acids extending from either end of the chain. For example, the chains may have a Ser-Lys sequence before th e "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 synthesizer. Journal of American Chemical Society, Vol. 85 Pages 2149-54(1963) . It is also possible to produce such peptides by genetic engineering techniques.
In accordance with another preferred embodiment, the peptide employed in conjunction with an antibiotic which inhibits DNA gyrase 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 preferably include the following basic peptide structure X12
- - R1 1-R1 1-R12-R13-R1 1-R14-R12-R1 1-
R14-R12-R11-R1 1-R1 1-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 a 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
12 is
(i) R12
( ii ) R14a-R12
( iii ) R1 1-R14a-R12
(iv) R14-R1 1-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 .
(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-R1 1-R14a-R12-R1 1-R1 1-R12-R13-
R11-R14-R12-R1 1-R1 1-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; wnerein 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 R1 7 are as hereinabove described, and n is 0 or 1, and each n may be the same or different.
The magainin peptides generally include at least fourteen amino acids and may include up to forty amino acids. A magainin peptide preferably has 22 or 23 amino acids. Accordingly, the hereinabove described basic peptide structures of a magainin peptide may include additional amino acids at the amino end or at the carboxyl end, or at both ends .
As representative examples of such magainin peptides, there may be mentioned peptides having the following primary sequence (expressed as a single letter code) as well as appropriate analogues and derivatives thereof :
(a) (NH2) GIGKFLHSAGKFGKAFVGEIMKS(OH) or (NH2)
(Magainin I)
(b) (NH2) GIGKFLHSAKKFGKAFVGEIMNS(OH) or (NH2)
(Magainin II)
(c) (NH2) GIGKFLHSAKKFGKAFVGEIMN(OH) or (NH2)
(Magainin III)
The following are examples of peptide derivatives or analogs of the basic structure:
(d) (NH2) IGKFLHSAKKFGKAFVGEIMNS(OH) or (NH2)
(e) (NH2) GKFLHSAKKFGKAFVGEIMNS(OH) or (NH2)
(f) (NH2) KFLHSAKKFGKAFVGEIMNS(OH) or (NH2)
Magainin peptides are described in Proc . Natl. Acad Sci. Vol. 84 pp 5449-53 (Aug. 87) . The term "magainin peptides" as used herein refers to the basic magainin structure as well as derivatives and analogs thereof , including but not limited to the representative derivatives or analogs.
In accordance with a further embodiment, the peptide employed in conjunction with an antibiotic which inhibits DNA gyrase 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-R1 1-R14-R14-R1 1-
R11-R14-R12-R1 1-R1 1-R12-R1 1-
R11-R11-R12- where R11 , R12 , R1 4, 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 followin g structure:
-Y14-X14- where X14 is as previously defined and
Y14 is
(i) R1 1;
(il) R14 -R11
where R11 and R14 are as previously defined.
For example, a PGLa peptide may also have the following structure:
-X14- Z14- where X14 is as previously defined; and Z14 is:
(I) R11 ; 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 X1 4; Y14 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
X16:
- -R11-R17-R12-Rn-R14-R18-R17-
R11-R14-R12-R1 1-R1 1-R12- R11- R11- R11- R12-(R15)n-R1 1 - , wherein
R11, R12 , R14, R15 and R17 are as Prevlously defined and R18 is glutamine or asparagine or a basic hydrophilic, or hydrophobic amino acid and, n is 0 or 1.
The XPF peptides generally include at least nineteen amino acids and may include up to forty amino acids . Accordingly, the hereinabove described basic peptide structure of XPF may include additional amino acids at the amino end, or at the carboxyl end or at both the amino and carboxyl ends.
Thus, for example, an XPF peptide may include the following structure:
-Y16-X16- where X16 is as previously defined and Y16 is
(i) R1 1 or
(ii) R14- R11
where R11 and R14 are 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 , Y 16 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 (single letter amino acid code) :
PGLa : GMASKAGAIAGKIAKVALKAL (NH2)
XPF : GWASKIGQTLGKIAKVGLKELIQPK
A review of XPF and PGLa can be found in Hoffman et al, EMBO J. 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 which inhibits DNA gyrase may be a CPF peptide or appropriate analogue or derviative thereof .
CPF peptides as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
The CPF peptide 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 are Ala, Cys, Phe, Gly, Ile, Leu, Met, Val, Trp, and Tyr.
The neutral hydrophilic amino acids are Asn, Gin, Ser, and Thr.
The basic hydrophilic amino acids are Lys, Arg, His, and ornithine.
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 Y3Q 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 follows:
-X30-Z30 wherein
X30 is the hereinabove defined basic peptide structure and Z30 is
(i) R21- '
(ii) R2l-R2 l- ;
(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 some of which have been described in the literature and comprise the following sequences (single letter amino acid code) :
( 1 ) GFGSFLGLALKAALKIG AN ALGGAPQQ
(2) GLASFLGKALKAGLKIGAHLLGGAPQQ
(3) GLASLLGKALKAGLKIGTHFLGGAPQQ
(4) GLASLLGKALKATLKIGTHFLGGAPQQ
(5) GFASFLGKALKAALKIGANMLGGTPQQ
(6) GFGSFLGKALKAALKIGANALGGAPQQ
(7) GFGSFLGKALKAALKIGANALGGSPQQ
(8) GFASFLGKALKAALKIGANLLGGTPQQ
A review of the CPF peptides can be found in Richter, K . , Egger, R. , and Kreil (1986) J. Biol. Chem. 261, 3676-3680; Wakabayashl, 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, L
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 analogue or derivative. The term CPF peptide includes the basic peptide structure as well as analogs or derivatives thereof .
In accordance with still another embodiment, the biologically active peptide may include the following basic strucutre X40:
[R41-R42-R42-R43-R41-R42-R42]n, wherein R41 is a basic hydrophilic amino acid, R42 is a hydrophobic amino acid, R43 is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5.
In one embodiment, such peptide may include the following structure :
Y40-X40, wherein X40 is as hereinabove described, and Y40 is:
(i) R42 ;
(ii) R42-R42 ;
(ill) R41-R42-R42;
(iv) R43-R41-R42-R42 ;
(V) R42-R43-R41-R42-R42 ; or
(vi) R42-R42-R43-R41-R42-R42 , wherein R41, R42 , and R43 are as hereinabove described
In accordance with another embodiment, such peptide may include the following structure:
X40- Z40, wherein X40 is as hereinabove described, and Z40 is:
(i) R41 ;
(ii) R41-R42 ;
(ill) R41-R42-R42 ;
(iv) R41-R42-R42-R43 ;
(v) R41-R42-R42-R43-R41; or
(vi) R41-R42-R42-R43-R43-R42 -
In accordance another embodiment, such peptide may include the following structure:
(Y40)a-X40-(Z40)b, wherein Y and Z are as previously defined, a is
0 or 1, and b is 0 or 1.
In one embodiment, n is 3, and most preferably the peptide is of the following structure as indicated by the single letter amino acid code:
[KIAGKIA]3.
In another embodiment, n is 2, and the peptide preferably is of the following structure as indicated by the single letter amino acid code:
KIA(KIAGKIA)2KIAG .
In accordance with yet another embodiment, the biologically active amphiphilic peptide may be a biologically active amphiphilic peptide including the following basic structure X50:
R41-R42-R42-R43-R4l-R42-R42-R41-R42-R42-R42-R41-R42-R42 ,
wherein R41, R42 and R43 are as hereinabove described.
In accordance with one embodiment, such peptide may include the following structure:
Y50-X50, wherein X50 is as hereinabove described, and Y50 is :
(i) R42 ;
(ii) R42-R42 ;
(iii) R41-R42-R42 ;
(iv) R43-R41-R42-R42 ;
(V) R42-R43-R41-R42-R42 ;
(vi) R42-R42-R43-R41-R42-R42 , or
(vϋ) R41-R42-R42-R43-R41-R42-R42 , wherein R41 , R42 and R43 are as hereinabove described.
In accordance with another embodiment, such peptide may include the following structure:
X50-Z50, wnerein X50 is as hereinabove described and Z50 is :
(i) R41;
(ii) R41-R42;
(iii) R41-R42-R42 ;
(iv) R41-R42-R42-R43;
(v) R41-R42-R42-R43-R41;
(vi) R41-R42-R42-R43-R41-R42 ; or
(vii) R41-R42-R42-R43-R41-R42-R42 , Wherein R41 , R42 and R43 are as hereinabove described.
In accordance with yet another embodiment the peptide may include the following structure:
( Y50)a-X50- (Z50)b, wherein X and Y are as previously defined, a is
0 or 1, and b is 0 or 1. In one embodiment, the peptide is of the following structural formula as indicated by the single letter amino acid code :
KLASKAGKIAGKIAKVALKAL.
In another embodiment, the peptide is of the following structural formula as indicated by the single letter amino acid code:
KIAGKIAKIAGOIAKIAGKIA.
In still another embodiment, the peptide employed in conjunction with an antibiotic which inhibits DNA gyrase is a cecropin. The cecroptns and analogs 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 cecropins includes the basic structure as well as analogues and derivatives.
In yet another embodiment, the peptide employed in conjunction with an antibiotic which inhibits DNA gyrase is a sarcotoxin . The sarcotoxins and analogs 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.
It is also contemplated that within the scope of the present invention, that each of the amino acid residues of the biologically active amphiphilic peptide structures hereinabove described is a D- amino acid residue or a glycine residue.
In another embodiment, an ion channel-forming protein may be used in conjunction with an antibiotic which inhibits DNA gyrase . Ion channel- forming proteins 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 (1987) . Perforin is described in Henkart, et al. J. Exp. Med. , 160: 75 (1984) , and in Podack, et al. , J. Exp . Med. 160:695 (1984) . The above articles are hereby incoroporated by reference.
The term ion channel-forming proteins includes the basic structures of the ion -forming proteins as well as analogues and derivatives.
The present invention will be further described with respect to the following example; however, the scope of the invention is not to be limited thereby.
Example 1
Approximately 1 - 5 × 10 colony forming units (CFU's) of P. aeruginosa strain 27853 or of P. aeruginosa strain 107 (which is gentamicin - resistant) dispersed in 100 ul of trypticase soy broth (TSB) were added to each of a series of test wells. Either Peptide 1, Peptide 2, or Peptide 3 was added to each test well in increasing amounts from 0.25 to 256 μg/ml in absence of or in the presence of 20% of the minimal inhibitory concentration (MIC) of ciprofloxacin. For purposes of this
example, Peptide 1 is amide-terminated Magainin II, Peptide 2 is of the following structural formula:
[KIAGKIA]3; and
Peptide 3 is of the following structural formula:
KLASKAGKIAGKIAKVALKAL.
The MIC of ciprofloxacin alone against P. aeruginosa strain 27853 was lμg/ml, and against P. aeruginosa strain 107 was 2 μg/ml. The MIC values for Peptides 1 , 2 and 3 , either alone or in combination with 20% of the MIC of ciprofloxacin are given in Table I below.
Table I
MIC (μg ./ml)
P.aeruginosa strain
27853 107
1. Peptide 1 alone >32 >32
2. Peptide 1 plus 32 32
20% MIC of
ciprofloxacin
3. Peptide 2 alone 32 32
4. Peptide 2 plus 16 16
20% MIC of
ciprofloxacin
5. Peptide 3 alone 16 32
6. Peptide 3 plus 8 8
20% MIC of
ciprofloxacin
Example 2
For Examples 2 through 4, microorganisms employed in the assays were grown according to the procedure described in Stutman, et al.
Antimicrobial Agents in Chemotherapy. Vol. 34, July 1990.
In Examples 2 through 4, organisms were subcultured on agar plates, and then grown in trypticase soy broth (TSB) , or Mueller-Hinton
broth. The final assays were then conducted in microtiter plates containing Mueller-Hinton broth 10 organisms were added to each well.
The minimal inhibitory concentrations (MIC's) of ciprofloxacin alone, of Peptide 1, as hereinabove described in Example 1 , alone, of ciprofloxacin when Peptide 1 was added (ciprofloxacin + Peptide 1) , and of Peptide 1 when ciprofloxacin was added, (Peptide 1 + ciprofloxacin) were tested against various isolates of strain MR- PSA Pseudomonas aeruginosa. The second compound (or "+ " compound) dose to establish synergy was the lowest dose of the synerglzing drug which alone lacked antibacterial effect and was at least a 50% lower dose than the MIC for the synerglzing agent alone. The results are given below in Table 2.
Table 2
MIC (ug/ml)
Ciprofloxacin Ciprofloxacin Peptide 1 Peptide 1
Isolate Alone +Peptide 1 Alone + Ciprofloxacin
1 <0.5 <0.5 32 8
2 <0.5 N/A >256 N/A
3 0.25 <0.25 32 8
4 1.0 <0.5 256 64
The minimal inhibitory concentrations, (MIC's) , according to the procedure of Example 2 , were tested against various isolates of strain MR- PSA, of Pseudomonas aeruginosa, except that Peptide 4 replaces Peptide 1. Peptide 4 has the following structural formula:
GIGKFLKSAKKFGKAFVKIMNS . The results are given below in Table 3.
Table 3
MIC f ug/ml)
Ciprofloxacin Ciprofloxacin Peptide 4 Peptide 4
Isolate# Alone + Peptide 4 Alone +ciprofloxacin
1 2 <0.5 32 8
2 0.25 0.25 4 4
3 0.25 0.25 8 8
4 0.063 0.25 8 8
5 0.25 0.063 8 4
6 4 <T0.5 32 8
7 0.125 0.25 16 16
8 0.5 0.5 16 16
9 2 <0.5 32 16
10 0.25 0.25 8 8
11 1 <0.125 64 0.5
12 2 <0.5 32 8
Example 4
The minimal inhibitory concentrations (MIC's) of Peptide 4 and ciprofloxacin, either alone or in combination with each other, according to the procedure described in Example 3, were tested against various isolates of Staphylococcus aureus. The results are given in Table 4 below .
Table 4
MIC (μg/ml)
Ciprofloxacin Ciprofloxacin Peptide 4 Peptide 4
Isolate* Alone + Peptide 4 Alone + Ciprofloxacin
1 1 0.125 32 8
2 0.25 0.25 16 16
3 0.5 0.125 32 2
4 >0.25 0.063 64 32
The peptide or protein and antibiotic which inhibits DNA gyrase, as 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. The peptide or protein and the antibiotic which inhibits DNA gyrase may be employed together in a single composition, or in separate compositions . Moreover, the antibiotic which inhibits DNA gyrase and the peptide or protein may be delivered or administered in different forms, for example, the
antibiotic which inhibits DNA gyrase may be administered systemically, while the peptide or protein may be administered topically.
The peptide or protein and/or antibiotic which inhibits DNA gyrase 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 which inhibits DNA gyrase 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, in particular bacteria .
The peptide(s) or protein(s) of the present invention may be administered to a host; in particular an animal, in an effective anti-microbial, in particular in an anti-bacterial amount, in conjunction with an antibiotic which inhibits DNA gyrase, for potentiating the activity of the peptide or protein.
As representative examples of administering the peptide or protein and antibiotic which inhibits DNA gyrase for topical or local administration, the peptide could be administered in an amount of up to about 1% weight to weight and the antibiotic which inhibits DNA gyrase delivered in an amount of about 50 mM (about 0.1%) . Alternatively, the antibiotic which inhibits DNA gyrase could be administered topically in conjunction with systemic administration of the peptide and/or protein. For example, the peptide or protein may be administered IV or IP to
achieve a serum dose of 100 micrograms per milliliter (10 milligrams per kilogram) in conjunction with a topical dose of antibiotic which inhibits DNA gyrase of from about 4 μg/ml to about 100 μg/ml.
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 other than as particularly described.
Claims
1. A process, comprising:
administering to a host at least one biologically active amphiphilic peptide and/or biologically active protein, said peptide or protein being an ion channel-forming peptide or protein; and
a antibiotic which inhibits DNA gyrase, said components being administered to inhibit growth of a target cell in a host.
2. The process of Claim 1 wherein the peptide is a magainin peptide.
3. The process of Claim 1 wherein the peptide includes the following basic structure X40:
[R41-R42-R42-R43-R41-R42-R42]n,
wherein R41 is a basic hydophillc amino acid, R42 is a hydrophobic amino acid, R43 is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5.
4. The process of Claim 1 wherein the peptide inlcudes the following basic structure X50: R41-R42-R42-R43-R41-R42-R42-R41-R42-R42-R42-
R41-R42-R42, whereln R41 ls a baslc hydrophilic amino acid, R42 is a hydrophobic amino acid, and R43 is a neutral hydrophilic or hydrophobic amino acid.
5. The process of Claim 1 wherein said antibiotic which inhibits DNA gyrase is a quinolone antibiotic.
6. The process of Claim 5 wherein said quinolone antibiotic is ciprofloxacin .
7. The process of Claim 1 wherein said antibiotic which inhibits DNA gyrase is nalidixic acid.
8. The process of Claim 1 wherein said antibiotic which inhibits DNA gyrase is oxolinic acid.
9. The process of Claim 1 wherein said antibiotic which inhibits DNA gyrase is cinoxacin.
10. A composition, comprising:
(a) at least one biologically active amphiphilic peptide and/or biologically active protein, said peptide or protein being an ion channel-forming peptide or protein; and
(b) an antibiotic which inhibits DNA gyrase.
11. The composition of Claim 10 wherein said components (a) and (b) are present in an amount to inhibit growth of a target cell.
12. The composition of Claim 10 wherein the peptide is a magainin peptide.
13. The composition of Claim 10 wherein the peptide includes the following basic strucutre X40:
[R41-R42-R42-R43-R41-R42-R42]n
wherein R41 is a basic hydrophilic amino acid, R42 is a hydrophobic amino acid, R43 is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5.
14. The composition of Claim 10 wherein the peptide inlcudes the following basic structure X50: R41-R42-R42-R43-R41-R42-R42-R41-R42-R42-R42-R41-R42-R42,
wherein R41 is a basic hydrophilic amino acid, R42 is a hydrophobic amino acid and R43 is a neutral hydrophilic or hydrophobic amino acid.
15. The composition of Claim 10 wherein said antibiotic which inhibits DNA gyrase is a quinolone antibiotic.
16. The composition of Claim 15 wherein said quinolone antibiotic is ciprofloxacin .
17. The composition of Claim 10 wherein said antibiotic which inhibits DNA gyrase is nalidixic acid.
18. The composition of Claim 10 wherein said antibiotic which inhibits DNA gyrase is oxolinic acid.
19. The composition of Claim 10 wherein said antibiotic which inhibits DNA gyrase is cinoxacin .
20. The composition of Claim 13 wherein said peptide is of the following sturcture:
[KIAGKIA]3.
21. The composition of Claim 14 wherein said peptide is of the following structure:
KLASKAGKIAGKIAKVALKAL
22. The composition of Claim 12 wherein said magainin peptide is Magainin II.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP91511793A JPH05507718A (en) | 1990-06-27 | 1991-06-20 | Compositions and therapeutic methods comprising bioactive peptides and antibiotics that inhibit DNA gyrase |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US54535690A | 1990-06-27 | 1990-06-27 | |
US545,356 | 1990-06-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1992000090A1 true WO1992000090A1 (en) | 1992-01-09 |
Family
ID=24175898
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1991/004453 WO1992000090A1 (en) | 1990-06-27 | 1991-06-20 | Composition and treatment with biologically active peptides and antibiotics which inhibit dna gyrase |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0559647A1 (en) |
JP (1) | JPH05507718A (en) |
CA (1) | CA2045073A1 (en) |
WO (1) | WO1992000090A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5654274A (en) * | 1992-06-01 | 1997-08-05 | Magainin Pharmaceuticals, Inc. | Biologically active peptides having N-terminal substitutions |
US5733872A (en) * | 1993-03-12 | 1998-03-31 | Xoma Corporation | Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof |
US6348445B1 (en) | 1992-06-01 | 2002-02-19 | Magainin Pharmaceuticals, Inc. | Biologically active peptides with reduced toxicity in animals and a method for preparing same |
Citations (6)
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 |
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 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2555174B2 (en) * | 1987-03-04 | 1996-11-20 | アメリカ合衆国 | Novel synthetic bioactive compounds and methods for producing bioactive effects |
-
1991
- 1991-06-20 JP JP91511793A patent/JPH05507718A/en active Pending
- 1991-06-20 WO PCT/US1991/004453 patent/WO1992000090A1/en not_active Application Discontinuation
- 1991-06-20 CA CA002045073A patent/CA2045073A1/en not_active Abandoned
- 1991-06-20 EP EP91912701A patent/EP0559647A1/en not_active Withdrawn
Patent Citations (6)
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 |
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 |
Non-Patent Citations (1)
Title |
---|
See also references of EP0559647A4 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5654274A (en) * | 1992-06-01 | 1997-08-05 | Magainin Pharmaceuticals, Inc. | Biologically active peptides having N-terminal substitutions |
US5686563A (en) * | 1992-06-01 | 1997-11-11 | Magainin Pharmaceuticals Inc. | Biologically active peptides having n-terminal substitutions |
US6348445B1 (en) | 1992-06-01 | 2002-02-19 | Magainin Pharmaceuticals, Inc. | Biologically active peptides with reduced toxicity in animals and a method for preparing same |
US5733872A (en) * | 1993-03-12 | 1998-03-31 | Xoma Corporation | Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof |
US5763567A (en) * | 1993-03-12 | 1998-06-09 | Xoma Corporation | Biologically active peptides from funcional domains of bactericidal/permeability-increasing protein and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
EP0559647A4 (en) | 1993-07-15 |
EP0559647A1 (en) | 1993-09-15 |
CA2045073A1 (en) | 1991-12-28 |
JPH05507718A (en) | 1993-11-04 |
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