WO1999003488A2 - Peptides biologiquement actifs a toxicite reduite chez l'animal et procedes d'elaboration - Google Patents

Peptides biologiquement actifs a toxicite reduite chez l'animal et procedes d'elaboration Download PDF

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Publication number
WO1999003488A2
WO1999003488A2 PCT/US1998/014610 US9814610W WO9903488A2 WO 1999003488 A2 WO1999003488 A2 WO 1999003488A2 US 9814610 W US9814610 W US 9814610W WO 9903488 A2 WO9903488 A2 WO 9903488A2
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WIPO (PCT)
Prior art keywords
peptide
amino acid
seq
biologically active
ion channel
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Application number
PCT/US1998/014610
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English (en)
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WO1999003488A3 (fr
Inventor
U. Prasad Kari
Taffy J. Williams
Michael Mclane
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Magainin Pharmaceuticals Inc.
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Publication date
Application filed by Magainin Pharmaceuticals Inc. filed Critical Magainin Pharmaceuticals Inc.
Priority to EP98933343A priority Critical patent/EP1001800A2/fr
Priority to AU83005/98A priority patent/AU8300598A/en
Priority to JP2000502785A priority patent/JP2001510164A/ja
Priority to CA002294518A priority patent/CA2294518A1/fr
Publication of WO1999003488A2 publication Critical patent/WO1999003488A2/fr
Publication of WO1999003488A3 publication Critical patent/WO1999003488A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to biologically active pepti ⁇ es. More particularly, this invention relates to biologically active peptides with reduced toxicity. Further, this invention relates to a method of modifying biologically active peptides, that are either N-terminally (ammo-terminal) substituted or unsubstituted, to reduce their toxicity in animals.
  • an unsubstituted biologically active peptide or protein there is provided an unsubstituted biologically active peptide or protein.
  • a N-terminal substituted peptide or protein having the formula:
  • X is a biologically active peptide or protein
  • N is a nitrogen atom
  • the peptide or protein is preferably an ion channel-forming peptide or protein.
  • T is a lipopk ⁇ ic moiety
  • W is another T group or hydrogen.
  • lipophilic means that the lipophilic moiety enhances the interaction of the peptide or protein with a lipid membrane, such as, for example, a cell membrane .
  • Lipophilic moieties which may be employed include, but are not limited to, any moiety which may be placed on the N-termmal of the peptide through a condensation reaction with nitrogen.
  • the lipophilic moiety T may be, for example, a carboxylic acid, a phosphoric acid, preferably an alkylphosphoric acid, a phosphonic acid, preferably an alkylphosphonic ac d, a sulfonic acid, preferably an alkylsulfonic acid, or an alkyl group.
  • T is:
  • R is a hydrocarbon having at least two and no more than 16 carbon atoms.
  • R is an alkyl group.
  • the alkyl group may be a straight chain or branched chain alkyl group, or a cycloalkyl group.
  • R may be CH 3 (CH 2 ) -, wherein n is from 1 to 14.
  • n is from 3 to 12, more preferably from 4 to 11, still more preferably from 6 to 11, and most preferably n is 6, whereby T is an octanoyl group.
  • R is an aromatic (including phenyl and naphthyl), or an alkyl aromatic group.
  • R may be
  • z is from 0 to 6.
  • z is 1 or 2
  • R is
  • n is from 1 to 5.
  • n is 1, whereby R is an ibuprofyl group.
  • T is:
  • HOOC - (CH 2 ) X -C wherein x is from 1 to 14.
  • x is 2, and T is a succinyl group.
  • T is:
  • CH 3 (CH 2 ) y -CH CH-CH-CH-NH-, OH wherein y is from 1 to 14.
  • y is 12, whereby T is a sphingosine group.
  • CH 3 (CH 2 ) y -CH CH-CH-CH-NH-C-(CH 2 ) X -C-, OH wherein x and y are hereinabove described.
  • x is 2, and y is 12.
  • W is hydrogen
  • Applicant has found that when biologically active peptides have substitutions at the N-terminal such as those hereinabove described, such peptides have increased biological activity against target cells, viruses, and virally-infected cells, as compared with unsubstituted peptides or peptides substituted at the N-terminal with an acetyl group. Applicant also has found that the N-terminal substitutions hereinabove described significantly increase the biological activity of "short" peptides, i.e., peptides having no more than 14 amino acid residues .
  • the biologically active peptides are not N-terminally substituted and have broad spectrum anti-tumor and anti-microbial activity.
  • the biologically active peptides or proteins of the present invention are preferably ion channel- forming peptides.
  • An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the permeability for ions across a natural or synthetic lipid membrane.
  • B. Christensen, et al., PNAS Vol. 85, pgs . 5072-5076 (July 1988) describes a 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 . This Christensen article is entirely incorporated herein by reference.
  • amphiphilic peptide or protein is a peptide or protein which includes both hydrophobic and hydrophilic peptide or protein regions .
  • the ion channel-forming peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water.
  • the structure of such peptides provides for flexibility of the peptide molecule.
  • Such peptides are capable of forming an alpha-helical structure. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself nto a rodlike structure.
  • such peptides have at least 7 ammo acids, and n many cases have at least 20 ammo acids. In most cases, such peptides ⁇ o not nave m excess of 40 am o acids.
  • the peptides and/or analogues cr derivatives tnereof 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, virus, or virally- fected cell.
  • a host for example a human or non-human animal
  • the peptides and/or analogues or derivatives thereof may be used as anti-microbial agents, anti-viral agents, anti-bacterial agents, anti-tumor agents, anti-parasitic agents, and spermicides, as well as agents exhibiting other bioactive functions.
  • anti-microbial means that the peptides or proteins of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes, such as bacteria, fungi, viruses, or the like.
  • anti-bacterial means that the peptides or proteins employed in the present invention produce effects adverse to the normal biological functions of bacteria, including death, destruction, or prevention of the growth or proliferation of the bacteria when contacted with the peptides or proteins .
  • antibiotic means that the peptides or proteins employed in the present invention produce effects a ⁇ verse to the normal biological functions of the non-host cell, tissue, or organism, including death, ⁇ estruction, or prevention of the growth or proliferation of the non-host cell, tissue, or organism when contacted with the pepti ⁇ es or proteins .
  • spermicidal as used herein means that the peptides or proteins employed m the present invention inhibit, prevent, or destroy the motility of sperm.
  • anti-fungal means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of fungi.
  • anti-viral means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of viruses, or of virally-infected cells.
  • anti-tumor means that the peptides or proteins inhibit the growth of or destroy tumors, including cancerous tumors.
  • anti-parasitic means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of parasites.
  • the peptides or proteins of the present invention have a broad range of potent anti-tumor and antibiotic activity against a plurality of tumor types and microorganisms, including gram- positive and gram-negative bacteria, fungi, protozoa, and the like, as well as parasites.
  • the peptides or proteins of the present invention allow a method for treating or controlling tumor growth and microbial infection caused by organisms which are sensitive to the peptides or proteins. Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an anti-tumor or anti-microbial amount of at least one of the peptides or proteins .
  • methods are provided for reducing the toxicity of unmodified peptides or of N-termmally modified peptides in a host without reducing the anti-tumor or anti-microbial activity of the peptides.
  • This method includes forming a methane sulfonate derivative or analogue of an unsubstituted peptides or N-termmal substituted peptide having the formula:
  • T is a biologically active peptide or protein, the peptide being an ion channel-forming peptide or protein
  • T is a lipophilic moiety or hydrogen.
  • the anti-tumor or anti-microbial activity of the methane sulfonate derivative or analogue of the unsubstituted or N-terminal substituted peptide is not reduced or is not significantly reduced as compared to the corresponding peptide not derivatized with a methane sulfonate group.
  • the phrase "not significantly reduced,” as used in this application, means that the methane sulfonate derivatives or analogues retain anti-tumor or anti-microbial activity of the underivatized compounds.
  • the methane sulfonate derivatives or analogues retain at least 50%, and preferably 75% or more, of the anti-tumor or anti-microbial activity of the underivatized compounds .
  • the methane sulfonate derivative can be formed by suspending a free base of the unsubstituted or N-terminal substituted peptide water, and then mixing the suspended peptide and at least C.5 equivalents of a sodium oisulfite-formaldehyde complex (or other suitable bisulfite-formaldehyde complex) for each free ammo group in the peptide. This reaction proceeds for a period of 10 minutes or more to produce the methane sulfonate derivative or analogue of the peptide.
  • the amount of the bisulfite-formaldehyde complex can be varied without departing from the invention.
  • the suspended free base is mixed with 0.5 to 10 equivalents of the bisulfite-formaldehyde complex for each free ammo group in the peptide.
  • the use of 1 to 5 equivalents is preferred, and 1.1 to 3 equivalents is particularly preferred.
  • the mixing period also can be varied widely.
  • the peptide free base and complex are mixed for a period of 10 minutes to 2 hours, or 10 minutes to 1 hour.
  • a mixing period in the range of from 15 minutes to 30 minutes is particularly preferred.
  • the starting free base of the unsubstituted peptide or N- term al substituted peptide can be prepared by neutralizing a salt of tr.e peptide. This neutralization can take place by treating the salt with a base solution, such as a sodium carbonate solution. After neutralization, the free base peptide, which may precipitate, can be isolated prior to suspending it in the water.
  • a base solution such as a sodium carbonate solution.
  • the methane sulfonate derivative or analogue of the peptide product can be recovered, e.g., by filtering. Additionally, this product can be lyophilized, if desired.
  • X is a biologically active peptide or protein
  • N is a nitrogen atom
  • T is a lipophilic moiety or hydrogen.
  • the peptide or protein is preferably an ion channel-forming peptide or protein.
  • the methane sulfonate derivative is formed by mixing the peptide salt with 12.5 equivalents of 30% formaldehyde solution and 6.25 equivalents of 1M sodium bicarbonate solution for eacn ammo group in the peptide.
  • the adduct of the peptide with formaldehyde precipitates and can be collected oy centrifugation of filtration.
  • the sodium methane sulfonate derivative is then formed by mixing the formaldehyde adduct with sodium metabisulfite .
  • the methane sulfonate analogue of the peptide is shown to have reduced toxicity in vivo, but retains its anti-tumor and anti-microbial activity.
  • antibiotics because of the antibiotic, anti-microbial, anti-viral, and anti-bacterial properties of the peptides or proteins, they may also be used as preservatives, sterilants, or disinfectants of materials susceptible to microbial or viral contamination.
  • the peptide or proteins and/or derivatives or analogues thereof may be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, a non-toxic buffer, or a physiological saline solution.
  • a non-toxic pharmaceutical carrier or vehicle such as a filler, a non-toxic buffer, or a physiological saline solution.
  • Such pharmaceutical compositions may be used topically or systemically ana may be in any suitable form such as a liquid, solid, semi-solid, miectable solution, tablet, ointment, lotion, paste, capsule, or the like.
  • the peptide or protein compositions may also be used in combination with ad uvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful microorganisms including protozoa, viruses, and the like, as well as by parasites.
  • the peptides or proteins of the present invention may be administered to a host, in particular a human or non-human animal, in an effective antiD_.ot ⁇ c and/or anti-tumor aid/or anti-fungai and/or anti-viral and/or anti-microbial and/or antibacterial and/or anti-parasitic and/or spermicidal amount.
  • composition accordance with the invention will contain an effective anti-microbial amount and/or an effective spermicidal amount and/or an effective anti-fungal amount and/or an effective anti-viral amount and/or an effective anti-tumor amount and/or an effective anti-parasitic amount and/or an effective antibiotic amount of one or more of the peptides or proteins of the present invention which have such activity.
  • the peptides or proteins may be administered by direct application of the peptides or proteins to the target cell, virus, or virally-infected cell, or indirectly applied through systemic administration.
  • the peptides or proteins of the present invention may also be employed in promoting or stimulating healing of a wound in a host .
  • wound healing includes various aspects of the wound healing process. These aspects include, but are not limited to, increased contraction of the wound, increased deposition of connective tissue, as evidenced by, for example, increased deposition of collagen in the wound, and increased tensile strength of the wound, i.e., the peptides or proteins increase wound breaking strength.
  • the peptides or proteins of the present invention may also be employed so as to reverse the inhibition of wound healing caused by conditions which depress or compromise the immune system.
  • the peptides or proteins of the present invention may be used in the treatment of external burns and to treat and/or prevent skin and burn infections.
  • the peptides or proteins may be used to treat skin and burn infections caused by organisms such as, but not limited to, P. aeruginosa and S. aureus .
  • the peptides or proteins 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, by fungi such as, but not limited to, C. albicans and A. fumiga tus, by parasites such as, but not limited to, A. castellam, or by viruses.
  • the peptides or proteins may also be effective in killing cysts, spores, or trophozoites of infection-causing organisms.
  • Such organisms include, but are not limited to Acanthamoeba , which form trophozoites or cysts, C. albicans, which form spores, and A. fumiga tus, which also form spores.
  • the peptides or proteins of the present invention may also be employed in the treatment of tumors.
  • tney are active against tumors that arise in tissues and organs such as, but not limited to, breast, lung, colon, rectum, cervix, ovaries prostate, stomach, as well as melanoma and leukemias. More specifically, they are active against non-small cell _.ung carcinomas and a ⁇ enocarcmomas of, for example, the breast, cervix, prostate, lung, colon, rectum, stomach, and ovaries.
  • the peptides and proteins of the present invention are active against tumors that are resistant to other anti-tumor agents. A significant reason for resistance, the removal of anti-tumor agents by the efflux pump, is not believed to apply to the peptides and proteins of the present invention which are believed to function via the unique mechanism of forming ion channels .
  • the peptides or proteins may also be administered to plants in an effective anti-microbial or anti-viral or anti-parasitic amount to prevent or treat microbial, viral, or parasitic contamination thereof.
  • the peptides or proteins of the present invention may also be employed in the treatment of serious lung infections.
  • the peptides or proteins may be used to treat lung infections caused by organisms such as, but not limited to, P. aeruginosa in cystic fibrosis patients.
  • the peptides or proteins are administered to a subject in need of treatment by an inhalation procedure.
  • the active peptide or protein ingredient can be delivered by inhalation using either a nebulizer, metered dose inhaler (MDI), or a dry powder inhaler (DPI) .
  • MDI metered dose inhaler
  • DPI dry powder inhaler
  • the active peptide or protein is present in the following amounts : for a nebulizer, a solution of between 5 and 200 mg/ml; for MDI or DPI, an amount between 0.5 and 45 mg .
  • the peptides or proteins also may be administered to a subject for treating sepsis, septic shock, and other related ailments, _r that such peptides neutralize bacterial endotoxins .
  • the pepti ⁇ es or proteins are positively charged, while, m general, the bacterial endotoxins are negatively charged.
  • the peptides or proteins are particularly useful that such compounds neutralize bacterial endotoxins without neutralizing essential proteins in plasma (such as heparin, for example) .
  • Treatment may mean complete elimination of a disease, ailment, or symptoms, or it may mean reducing, suppressing, or ameliorating the severity of the disease, ailment, or symptoms.
  • the peptides or proteins when used in topical compositions, are generally present in an amount of at least 0.1%, by weight. In most cases, it is not necessary to employ the peptide in an amount greater than 2.0%, by weight.
  • the active peptide or protein is present in an amount to achieve a serum level of the peptide of at least about 5 ⁇ g/ml.
  • the serum level cf peptide or protein need not exceed 500 ⁇ g/ml.
  • a preferred serum level is about 100 ⁇ g/ml.
  • Such serum levels may be achieved by incorporating the peptide or protein in a composition to be administered systemically at a dose of from 1 to about 10 mg/kg.
  • the peptide (s) or protein (s) need not be administered at a dose exceeding 100 mg/kg.
  • the peptides or proteins may be produced by known techniques and obtained in substantially pure form.
  • the peptides may be synthesized on an automatic peptide synthesizer. Journal of the American Chemical Society, Vol. 85, pgs. 2149-54 (1963) (which article is entirely incorporated herein by reference) . It also is possible to produce such peptides or proteins by genetic engineering techniques.
  • the codons encoding specific amino acids are known to those skilled in the art, and therefore, DNA encoding the peptides may be constructed by appropriate techniques, and one may clone such DNA into an appropriate expression vehicle (e.g., a plasmid) which is transfected into an appropriate organism for expression of the peptide or protein.
  • an appropriate expression vehicle e.g., a plasmid
  • the N-terminal (NH 2 or amino terminal) of the peptide is reacted such that the lipophilic moiety is attached to the N-terminal of the peptide.
  • the reaction may be a condensation reaction with an amine.
  • R - C - the N-termmal is reacted with a carboxylic acid of the formula R-COOH, wherein R is a hydrocarbon having at least 2 carbon atoms .
  • the reaction may be carried out in the presence of a coupling agent, such as, for example, DCC, or DIC, and HOBT, or in the presence of an acid chloride.
  • a coupling agent such as, for example, DCC, or DIC, and HOBT
  • X is a peptide which is a basic (positively charged) polypeptide having at least sixteen amino acids, wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids.
  • 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 am o acids, and the amino acids between pairs of hydrophobic am o acids may or may not be hydrophilic.
  • the hydrophilic ammo 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 ammo acids) and generally no greater than four amino acids, and the ammo acids between pairs of hydrophilic am o 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 ammo acids. Two of the four ammo acids in each group are hydrophobic ammo acids, and two of the four ammo acids in each group are hydrophilic, with at least one of the hydrophilic ammo acids in each group being a basic hydrophilic ammo acid and the other being a basic or neutral hydropnilic ammo acid.
  • the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie, Leu, Met, Pro, Val, Trp, Tyr, norleuc e (Nle) , norvaline (Nva), and cyclohexylalan e (Cha) .
  • the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gin, Ser, Thr, and homoserine (Hse) .
  • the basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginone (Har) , 2, 4-diam ⁇ nobutyr ⁇ c acid (Dbu), and p-aminophenylalanme .
  • 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 ammo 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 ammo 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 ammo acids, ana no greater than 50 ammo 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 ammo acids extending from either or both ends of the noted groups forming tne polypeptide chain and/or there may be am o aci ⁇ s between one or more of the at least four groups and still remain within the scope of the invention.
  • the groups of am o acids may be repeating groups of amino acids, or the ammo acids in the various groups may vary, provided tnat m each group of the at least four groups of ammo acids there are two hydrophobic and two hydrophilic ammo acids as here aoove noted.
  • the biologically active polypeptide may comprise a chain including at least four groups of amino acids, each containing four ammo acids . Two of the four ammo 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:
  • Y x 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-B-;
  • Y 3 is -C, -C-D, -C-D-A
  • Xpurchase 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 does not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted groups of four amino acids are not spaced from each other.
  • the peptide may have amino acids extending from either end of the chain.
  • the chains may have a Ser-Lys sequence before the "Ala” end, and/or an Ala-Phe sequence after the "Lys" end.
  • Other amino acid sequences may also be attached to the "Ala” and/or the "Lys" end.
  • the chain may have, for example, a C-D sequence before the first A- B-C-D group.
  • other amino acid sequences may be attached to the "A" and/or the "D" end of one of these polypeptide chains.
  • X is a magainin peptide .
  • a magainin peptide is either a magainin such as magainin I, II, or III, or an analogue or derivative thereof.
  • the magainin peptides preferably include the following basic peptide structure X 12 : Rl4 - Rl2 - R: i - Rl l - l l - R l 4a ⁇ ( R 1 5 ) n- f i a _ Rl 4 ⁇ ⁇ wherein R u 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, 4a are hydrophobic or basic hydrophilic amino acids; R 15 is glutamic acid or aspartic acid; and 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 u , 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; or
  • R, 6 -R where R 1 is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid.
  • R 17 is a basic 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 :
  • the magainin peptide may also include the following structure : -X 13 - Z 13 - , wherein X 13 is the hereinabove described basic peptide s gagture and Z 13 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 having the following primary sequences as given in the accompanying sequence listing, as well as appropriate analogues and derivatives thereof:
  • Magainin peptides are described in Proc. Na tl . Acad Sci . Vol. 84 pp. 5449-53 (Aug. 87), which article is entirely incorporated herein by reference.
  • 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.
  • X may be a PGLa peptide or an XPF peptide.
  • a PGLa peptide is either PGLa or an analogue or derivative thereof.
  • the PGLa peptides preferably include the following basic peptide structure X 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 ends .
  • a PGLa peptide may have the following structure : -Y 14 -X 14 - where X 14 is as previously defined and
  • a PGLa peptide may also have the following structure :
  • a PGLa peptide may also have the following structure: where X 14 , Y 14 , and Z 14 are as previously defined, a is 0 or
  • An X?F peptide is either XPF or an analogue or derivative thereof.
  • the XPF peptides preferably include the following basic peptide structure X 16 : u- i7 — i2- ⁇ - i -Ri ⁇ — n- Ru — Ri4 - i2 _ Ru- ⁇ - ⁇ :- u ⁇ ⁇ Ru -R 12 - ( R 15 ) ⁇ -Ru ⁇ - ' wherein R : ⁇ , R 12 , R 14 , R 15 , and R 17 are as previously defined; R l ⁇ is glutamine, asparagine, a basic hydrophilic amino acid, or a 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 ammo acids. Accordingly, the hereinabove described basic peptide structure of XPF may include additional ammo 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:
  • X 16 is as previously defined and Z 16 is: (i) R__; ( ii ) R_ ⁇ -R_ fashion; (iii) R n -R 18 -Prol ⁇ ne; or
  • An XPF peptide may also have the following structure: where X 16 , Y 16 , and Z 16 are as previously defined, a is 0 or
  • XPF or PGLa peptides which are characterized by the following primary amino acid sequences, as given in the accompanying sequence listing:
  • X is a CPF peptide or an appropriate analogue or derivative thereof.
  • CPF peptides as well as analogues and derivatives thereof, are herein sometimes referred to collectively as CPF peptides.
  • the CPF peptide may be one which includes the following basic peptide structure X 20 :
  • 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, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle) , norvaline (Nva) , and cyclohexylalanine (Cha) .
  • the neutral hydrophilic amino acids are Asn, Gin, Ser, Thr, and homoserine (Hse) .
  • the basic hydrophilic amino acids are Lys, Arg, His, Orn, homoarginine (Har), 2, 4-diaminobutyric acid (Dbu), and p-aminophenylal
  • the CPF peptide may include only the hereinabove noted amino acids or may include additional amino acids at the amino and/or carboxyl ends or both the amino and carboxyl ends . In general, the peptide does not include more than 40 amino acids.
  • the CPF peptides including the above basic structure preferably have from 1 to 4 additional amino acids at the amino end.
  • Such preferred peptides may be represented by the structural formula: 0- ⁇ 20 — wherein X 20 is the hereinabove described basic peptide structure and Y 20 is
  • the carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids.
  • the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows: -X 20 -Z 20 , wherein
  • X 20 is the hereinabove defined basic peptide structure and Z 20 is
  • Preferred peptides may be represented by the following structural formula
  • CPF peptides which may be employed, some of which have been described m the literature, include the following sequences as given in the accompanying sequence listing:
  • X is a peptide which includes one of the following basic structures X 31 through X 37 wherein:
  • X 37 IS - [ 32-R31- 32- 32 ⁇ R33 _ R31 ⁇ 32J ⁇ n'
  • R 3 is a basic hydrophilic amino acid
  • R 32 is a hydrophobic amino acid
  • R 33 is a neutral hydrophilic, basic hydrophilic, or hydrophobic amino acid
  • n is from 1 to 5.
  • the basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4- diaminobutyric acid (Dbu) , and p-aminophenylalanine.
  • the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, He, Leu, Met, Pro, 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, Thr, and homoserine (Hse) .
  • the peptide when the peptide includes the structure X 31 , 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:
  • the peptide may include the following structure:
  • Y 32 -X 32 wherein X 32 is as hereinabove described, and Y 32 is:
  • the peptide when the peptide includes the structure X 32 , the peptide may include the following structure:
  • X 32 -Z 32 wherein X 32 is as hereinabove described, and Z 32 is:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 33 , the peptide may include the following structure :
  • Y 33 -X 33 wherein X 33 is as hereinabove described, ana Y_ is:
  • the peptide when tne peptme includes the structure X 33 , the peptide may include the following structure :
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 34 , the peptide may include the following structure :
  • Y 34 -X 34 wherein X 34 is as hereinabove described, and Y 34 is:
  • the peptide when the peptide includes the structure X 34 , the peptide may include the following structure :
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 35 , the peptide may include the following structure :
  • Y 35 -X 35 wherein X 35 is as hereinabove described, and Y 35 is:
  • the peptide when the peptide includes the structure X 35 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 36 , the peptide may include the following structure :
  • Y 36 -X 36 wherein X 36 is as hereinabove described, and Y 36 is:
  • 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 include the structure Y 37 -X 37 , wherein X 37 is as hereinabove described, and Y is:
  • the peptide when the peptide includes the structure X 37 , the peptide may include the following structure :
  • X 37 -Z 37 wherein X 37 is as hereinabove described, and Z 37 is: (i) R 32 ; (iii) R 32 -R 3 ⁇ -R 3 ;
  • the peptide may include the following structure:
  • n 3 or 3
  • Xaa is p-aminophenylalanine .
  • X is a peptide which includes the following basic structure X 40 :
  • the peptide may include the following structure:
  • X 40 is as hereinabove described, and Y 40 is: ( i ) R32 ; (ill) R 34 -R 32 -R 32 (iv) R 33 -R 34 -R 32 -R 32 ;
  • X is a peptide which includes the following structure:
  • X 40 -Z. 7 wherein X 40 is as hereinabove described ana Z 40 is:
  • the peptide may include the following structure:
  • the peptide has the following structural formula as given in the accompanying sequence listing:
  • the peptide has the following structural formula as given in the accompanying sequence listing:
  • the peptide has one of the following structural formulae as given in the accompanying sequence listing:
  • X is a peptide which includes one of the following structural formulae: (i) - (Lys He Ala Lys Lys He Ala) n -, (ii)- (Lys Phe Ala Lys Lys Phe Ala) n -, and
  • n is from 1 to 5.
  • n is 3, and the peptide has one of the following structural formulae:
  • the X is a peptide which is selected from tne group consisting of tr.e following structural formulae as given in the accompanying sequence listing:
  • X is a cecropin or sarcotox n.
  • cecropin includes the basic structure as well as analogues and derivatives thereof. The cecropins and analogues and derivatives thereof are described in Ann . Rev. Microbiol . , 1987, Vol. 41, pages 103-26, in particular page 108, and in Christensen, et al., PNAS, Vol. 85, pgs . 5072-76, which are hereby incorporated by reference.
  • sarcotoxin includes the basic materials as well as analogues and derivatives thereof.
  • the sarcotoxins and analogues and derivatives thereof are described in Molecular Entomology, pages 369-78, in particular page 375, Alan R. Liss, Inc. (1987), which is hereby incorporated by reference.
  • X is melittin or an analogue or derivative thereof.
  • Melittin is an amphipathic peptide consisting of 26 amino acid residues, and is isolated from honeybee (Apis mellifera) venom. Habermann, et al., Hoppe-
  • X is an amphiphilic peptide which is a hybrid of a cecropin peptide and a melittin peptide or an analogue thereof.
  • Such hybrid peptides are described in U.S.
  • R 41 is a hydrophobic amino acid
  • R 42 is a basic hydrophilic or neutral hydrophilic amino acid.
  • the peptide includes the basic structure Y 5D -X 50 , wherein X 50 is as hereinabove described and Y 50 is :
  • R 41 is leucine.
  • R 42 is lysine.
  • Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:
  • X is an amphiphilic peptide which includes the following basic structure X 52 : R 4 2 — R 4 ⁇ -R42 — R2 — 4i — R1"" 2 — R 4 2 — R41""R42”” 2 wherein Rj is a hydrophobic amino acid, and R 42 is a basic hydrophilic or neutral hydrophilic amino acid.
  • R 41 is leucine. In another embodiment, R 42 is lysine.
  • the peptide includes the basic structure Y 52 -X 52, wherein X 52 is as hereinabove described, and Y 52 is:
  • the peptide may have the following structure :
  • the peptide includes the basic structure X 52 -Z 52 , wherein X 52 is as hereinabove described, and Z 52 is :
  • R1-R1- 42-R2 (iv) R1-R1- 42-R2; or (v) R i ⁇ R 4i ⁇ R 2 ⁇ R 2 ⁇ R4i ' wherein R 41 and R 42 are as hereinabove described .
  • the peptide may have the following structure :
  • the peptide may include the structure :
  • X is a biologically active amphiphilic peptide which includes the following basic structure X 54 : 41 ⁇ R42 _ R42-R 1- 4l" ⁇ R42- 2-R41-R 2-R42-R41 _ R 1 _ R 2 ⁇ R42-R42- 43 wherein R 41 and R 42 are as hereinabove described, and R 43 is a neutral hydrophilic amino acid.
  • the peptide may have the following structure:
  • the peptide may have the following structure:
  • X is a biologically active amphiphilic peptide which includes the following basic structure X 56 : R4i- 42 ⁇ R2 ⁇ R4i ⁇ R i ⁇ R2 ⁇ R42 ⁇ R4i- i-R42 ⁇ R2 ⁇ 44f wherein R 41 and R 42 are as hereinabove described, and R 44 is a neutral hydrophilic amino acid or proline.
  • the peptide may include the structure: X 56 -Z 56 , wherein X 56 is as hereinabove described, and Z 56 is: (I) -R 42 ;
  • the peptide may have one of the following structures:
  • X is a biologically active amphiphilic peptide which includes the following basic structure X 58 :
  • the peptide includes the structure X 58 ⁇ Z 58 , wherein X 58 is as hereinabove described, and Z 58 is: (ii) -R 41 -R 45 ; (in) -R 1 —R 5 -R 45 ; (iv) -R 41 -R 45 -R 45 -R 43 ;
  • the peptide has the following structure :
  • X is a biologically active amphiphilic peptide which includes the following basic structure X 60 :
  • the peptide may have the following structure:
  • X is a peptide which includes the following basic structure X 62 :
  • R 41 and R 42 are as hereinabove described.
  • the peptide includes the following structure Y 62 - X 62 , where X 62 is as hereinabove described, and Y 62 s:
  • the peptide includes the structure x 62 ⁇ z 62 / wherein X 62 is as hereinabove described, and Z 62 is:
  • the peptide has the structure ( ⁇ 62>a ⁇ x 62 ⁇ ( z 62 ) b f wherein X 62 , Y 62 , and Z 62 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
  • X is a peptide having the following structural formula :
  • X is a biologically active amphiphilic peptide including the following basic structure
  • R 41 and R 42 are as hereinabove described.
  • the peptide may include the structure Y 6 ⁇ Xe 4 , wherein X 64 is as hereinabove described, and Y 64 is:
  • the peptide may include the structure X 64 -Z 64 , wherein X 64 is as hereinabove described, and Z 64 is:
  • the peptide has the structure:
  • X is a biologically active amphipnilic peptide including the following basic structure 66 :
  • R 41 and R 42 are hereinabove described and R 46 is glutamic acid.
  • a representative example of such a peptide is the following:
  • X is a biologically active amphiphilic peptide including the following basic structure
  • the peptide includes the following basic structure Y 68 -X 68 , wherein X 68 is as hereinabove described, and Y 68 is R 41 (defined above) .
  • X is a biologically active amphiphili c peptide including the following basic structure X 70 .
  • R 4 ⁇ — 42 _ R 42 — R 4 ⁇ — R 4 1- R 2 ⁇ 42 ⁇ ⁇ ii — 2 — 42- i — 41 ⁇ ' n ⁇ rein R 41 and R 42 are hereinabove described .
  • a representative example of such a peptide has the following structure :
  • X is a biologically active amphiphilic peptide including the following basic structure X 72 :
  • R 4 , and R 42 are hereinabove described, and R.-. is aspartic acid.
  • R.-. is aspartic acid.
  • a representative example of such a peptide has the following structure :
  • X is a biologically active amphiphilic peptide having the following structure:
  • X is a biologically active amphiphilic peptide including the following structure X 74 : R2-R1-R42-R41- 1-R42-R42-R41-R6-R2-R1/ wherein R 41 , R 42 , and R 46 are hereinabove described.
  • X 74 R2-R1-R42-R41- 1-R42-R42-R41-R6-R2-R1/ wherein R 41 , R 42 , and R 46 are hereinabove described.
  • a representative example of such a peptide has the following structure:
  • X is a biologically active amphiphilic peptide including the following structure X 76 :
  • R 41 and R 42 are hereinabove described.
  • the peptide includes the structure Y 76 -X-, 6 -, wherein X 76 is as hereinabove described, and Y- is:
  • the peptide includes the structure X-. 6 -Z 76 , wherein X-, 6 is as hereinabove described, and Z 16 is:
  • R 48 -R 41 -R 42 wherein R 41 and R 42 are as hereinabove described, and R 48 is a basic hydrophilic, neutral hydrophilic, or hydrophobic amino acid.
  • the peptide has the following structural formula: (Y 76 ) a -X 76 - (Z 76 ) b , wherein X 76 , Y 76 , and Z 6 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
  • X is a biologically active amphiphilic peptide including the following structural formula X 78 :
  • X has the following structure :
  • X is a biologically active amphiphilic peptide including the following structural formula
  • R 41 , R 42 , and R 46 are as hereinabove described.
  • a representative example of such a peptide has the following structure:
  • X is an ion channel-forming peptide or protein.
  • Ion channel-forming proteins or peptides which may be employed include defensins, also known as human neutrophil anti-microbial 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 anti-microbial 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 . , J
  • 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 each are entirely incorporated herein by reference.
  • ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as analogues and derivatives.
  • each of the amino acid residues of the peptides or proteins may be a D- amino acid or glycine.
  • the scope of this particular embodiment is not to be limited to any theoretical reasoning, it is believed that the above-mentioned peptides or proteins, when consisting entirely of D-amino acid or glycine residues, may have increased resistance to proteolytic enzymes while retaining their activity. Such peptides thus may be administered orally.
  • all of the amino acid residues may be D-amino acid or glycine residues, or L-amino acid or glycine residues. It is also to be understood that the peptides or proteins may be administered in combination with one another.
  • N-terminal substituted peptides or proteins of the present invention may be employed in combination with an ion having pharmacological properties for the purposes hereinabove described.
  • An ion having pharmacological properties is one which, when introduced into a target cell, virus, or virally-mfected cell, inhibits and/or prevents and/or destroys the growth of the target cell, virus, or virally-infected cell.
  • Such an ion having pharmacological properties is one which, in the absence of an ion channel forming peptide, is unable to cross a natural or synthetic lipid membrane, in particular a cell or virus membrane, m sufficient amounts to affect a cell or virus adversely.
  • the peptide or protein and on having pharmacological properties may be administere ⁇ as a single composition or m separate compositions, and the single or separate compositions may include additional materials, actives, and/or inactives, in addition to the peptide or protein and ion having pharmacological properties.
  • ions having pharmacological properties there may be mentioned fluoride, peroxide, bicarbonate, silver, zinc, mercury, arsenic, copper, platinum, antimony, gold, thallium, nickel, selenium, bismuth, and cadmium ions.
  • the peptide or protein and the ion having pharmacological properties are employed in amounts effective to inhibit and/or prevent and/or destroy the growth of the target cell, virus, or virally-mfected cell.
  • the ion potentiates the action of the peptide, i.e., the amount of ion is effective to reduce the maximum effective concentration of the peptide or protein for inhibiting growth of a target cell, virus, or virally-mfected cell.
  • the ion having pharmacological properties when used topically, is generally employed in a concentration of from 0.05% to 2.0%. When used systemically, the ion is generally employed in an amount of from 1 to 10 mg . per kg. of host weight. Peptide or protein dosages may be within the ranges hereinabove described.
  • the peptide or protein and ion having pharmacological properties may be delivered or administere ⁇ m different forms.
  • the ion may be administere ⁇ orally, while the peptide may be administered by IV or IP.
  • the peptide could be administered in an amount of up to about 1% weight to weight and the ion delivered in an amount of about 50mM (about 0.1%).
  • the ion, in the form of a salt such as sodium fluoride could be administered orally in conjunction with systemic administration of the peptide or protein.
  • the peptide or protein may be administered IV or IP to achieve a serum dose of 100 icrograms per milliliter (10 milligrams per kilogram) in conjunction with an oral dose of ion, in particular, sodium fluoride, of 10 meq per kilogram.
  • the peptides or proteins of the present invention may be administered to a host in combination with an antibiotic selected from the class consisting of bacitracins, gramacidin, polymyxin, vancomycm, teichoplanm, aminoglycosides, hydrophobic antibiotics, penicillin, monobactams, or derivatives or analogues tnereof.
  • an antibiotic selected from the class consisting of bacitracins, gramacidin, polymyxin, vancomycm, teichoplanm, aminoglycosides, hydrophobic antibiotics, penicillin, monobactams, or derivatives or analogues tnereof.
  • the bacitracins are a group of polypeptide antibiotics.
  • a preferred bacitrac is bacitracin A.
  • Aminoglycoside antibiotics include tobramycm, kanamycin, amikac , the gentamicine (e.g., gentamicm C ⁇ f gentamicm C 2 , gentamicin C la ) , netilmicin, and derivatives and analogues thereof.
  • the preferred aminoglycosides are tobramycm and the gentamic s.
  • the aminoglycosides, and the bacitracins hereinabove described, tend to be hydrophilic and water- soluble .
  • Penicillins which may be employed include, but are not limited to, benzyl penicillin, ampicillin, methicillin (dimethoxyphenyl penicillin) , ticaricillin, penicillin V (phenoxymethyl penicillin) , oxacillin, cloxacillin, dicloxacillin, flucloxacillin, amoxicillin, and amidinocillin.
  • Preferred penicillins which may be employed are benzyl penicillin and ampicillin.
  • a preferred monobactam which may be employed is aztreonam.
  • hydrophobic antibiotics which may be used in the present invention, there may be mentioned macrolides, such as erythromycin, roxythromycin, clarithromycm, etc.; 9-N-alkyl derivatives of erythromycin; midecamycm acetate; azithromycm; flurithromycin; ⁇ fabutm; rokitamycin; a 6-0-methyl erythromycin A known as TE-031
  • rifamycin carbenicilllin, and nafcillin may be employed as well .
  • antibiotics which may be used are antibiotics which are 50-S ribosome inhibitors such as lincomycin; clindamycm; and chloramphenicol; etc.; and antibiotics which have a large lipid like lactone ring, such as mystatin; pimaricin, etc.
  • the peptide or protein and antibiotic may be administered by direct administration to a target cell by systemic or topical administration to a host which includes the target cell, in order to prevent, destroy, or inhibit the growth of a target cell.
  • Target cells whose growth may be prevented, inhibited, or destroyed by the administration of the peptides and antibiotic include Gram-positive and Gram-negative bacteria, as well as fungal cells.
  • the antibiotic such as those hereinabove described, or derivatives or analogues thereof, when used topically, is generally employed in a concentration of about 0.1% to about 10% (by weight) .
  • the antibiotic or derivative or analogue thereof when used systemically, is generally employed in an amount of from 1.25 mg. to about 45 mg. per kg. of host weight per day.
  • Peptide or protein dosages may be those as hereinabove described.
  • the peptide or protein could be administered in an amount of from 0.1% to about 10% weight to weight, and the antibiotic is delivered in an amount of from about 0.1% to about 10% weight to weight.
  • the peptides or proteins of the present invention may be administered in combination with an anti-parasitic agent or an anti-fungal agent .
  • Anti-parasitic agents which may be employed include, but are not limited to, anti-protozoan agents.
  • Examples of specific anti-parasitic agents which may be employed include, but are not limited to, pentamidine lsethionate, and propamidine lsethionate (Brolene) .
  • Anti-fungal agents which may be employed include, but are not limited to, ketoconazole . It is also to be understoo ⁇ that certain anti-parasitic agents may also have anti-fungal activity, and that anti-fungal agents may have anti-parasitic activity .
  • the peptides or proteins of the present invention may be administered in combination with an antibiotic which inhibits DNA gyrase, which is an enzyme involved in the formation of bonds between individual coiling strands of replicating bacterial DNA.
  • DNA gyrase is necessary for tr.e 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 qumolone antibiotics which include ciprofloxacin, norfloxacin, ofloxacin, enoxacin, pefloxacin, lomefloxacin, fleroxacin, tosulfoxacin, temafloxac , and rufloxacin.
  • Figure 1 is a graphical comparison of the maximum tolerated dose of Compound 843 and its methane sulfonate derivative, Compound 1324 (see Example 8, Table VII).
  • Figure 2 is a survival curve for A-549 human lung carcinoma cells treated with MSI-78 (SEQ ID NO: 154-NH,) and MSI-1858(SEQ ID NO: 154-NH 2 methane sulfonate).
  • Figure 3 is a survival curve for A-549 human lung carcinoma cells treated with MSI-344(SEQ ID NO: 154-OH) and MSI-1857(SEQ ID NO: 154-OH methane sulfonate).
  • Figure 4 is a survival curve for MDA-435 human breast carcinoma cells treated with MSI-78 (SEQ ID NO: 154-NH : ) and MSI-1858(SEQ ID NO: 154-NH 2 methane sulfonate).
  • Figure 5 is a survival curve for MDA-435 human breast carcinoma ceils treated with MSI-344(SEQ ID NO: 154-OH) and MSI-1857(SEQ ID NO: 154-OH methane sulfonate).
  • Figure 6 is a survival curve for K-562 human chronic myelogenous leukemia cells treated with MSI-78 (SEQ ID NO: 154- NH 2 ) and MSI-1858(SEQ ID NO: 154-NH 2 methane sulfonate).
  • Figure 7 is a survival curve for K-562 human chronic myelogenous leukemia cells treated with MSI-344(SEQ ID NO: 154-OH) and MSI-1857 (SEQ ID NO: 154-OH methane sulfonate).
  • Figure 8 is a survival curve for WM 1617 human melanoma cells treated with MSI-78 (SEQ ID NO: 154-NH 2 ) and MSI-1858 (SEQ ID NO: 154-NH 2 methane sulfonate) .
  • Figure 9 is a survival curve for WM 1617 human melanoma cells treated with MSI-344(SEQ ID NO: 154-OH) and MSI-1857(SEQ ID NO: 154-OH methane sulfonate) .
  • Figure 10 is a survival curve for CD-I mice administered a single iv dose of MSI-344 (SEQ ID NO: 154-OH) or MSI-1857(SEQ ID NO: 154-OH methane sulfonate) .
  • Figure 11 is a survival curve for CD-I mice administered a single ip dose of MSI-344(SEQ ID NO: 154-OH) or MSI-1857(SEQ ID NO: 154-OH methane sulfonate) .
  • Figure 12 is a survival curve for SCID mice dosed twice a week with either MSI-344(SEQ ID NO: 154-OH) or MSI-1857 (SEQ ID NO: 154-OH methane sulfonate) .
  • Figure 13 is a body weight curve for SCID mice dosed three times a week with either MSI-344(SEQ ID NO: 154-OH) or MSI-1857(SEQ ID NO: 154-OH methane sulfonate).
  • Figure 14 is a body weight curve for SCID mice dosed twice a week with either MSI-344 (SEQ ID NO: 154-OH) or MSI- 1857 (SEQ ID NO: 154-OH methane sulfonate).
  • Figure 15 is a standard curve relating antibacterial activity (zone diameter) to the concentration of MSI-1324 (OCT- SEQ ID NO: 143-NH 2 methane sulfonate).
  • Figure 16 shows the concentration of MSI-1324 ) OCT-SEQ ID NO: 143-NH 2 methane sulfonate) in plasma after a single iv dose .
  • Table I which follows, indicates the Minimal Inhibitory Concentration (MIC) in ⁇ g/ml of various peptides against S . aureus strain ATCC 25923 (S), P. aeruginosa strain ATCC 27853 (P), E . coli ATCC strain 25922(E), and C. albicans (CA) .
  • a "D” indicates that each ammo acid residue is a D-ammo acid residue or a glycine residue.
  • the peptides are unsubstituted at the N-terminal; substituted with an acetyl group at the N- terminal, as indicated by Ac-; substituted with an octanoyl group at the N-terminal, as indicated by Oct-; substituted with spinogos e, as indicated by Sph-; substituted with a succ yl group, as indicated by Sue-; substituted with a hexanoyl group, as indicated by Hex-; substituted with a heptanoyl group, as indicated by Hep-; substituted with a valeryl group, as indicated by Val-; substituted with a my ⁇ stryl group, as indicated by Myr-; or substituted with an ibuprofyl group, as indicated by Ibu-.
  • the stock peptide solution was diluted in serial dilutions (1:2) down the wells of a microtiter plate so that the final concentrations of peptides in the wells were 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128 and 256 ⁇ g/ml. 1-5 x 10 5 CFUs/ml of either S . aureus ATCC 25923, E . coli ATCC 25922, P. aeruginosa ATCC 27853, or C. albicans, were added to the wells in full strength Mueller Hinton broth (BBL 11443) from a mid- log culture. The inoculum was standardized spectrophotometrically at 600 nm and was verified by colony counts.
  • MIC minimal inhibitory concentration
  • cultures were taken from overnight (24 hour) broth cultures and diluted in fresh BHI broth (plus hemin plus vitamin I to deliver 1 x 10 6 colony-formmg units (CFUs)/ml m each microtiter test well.
  • NCLS National Committee for Clinical Laboratory Standards
  • Microtiter plates (Corning, Corning, NY) were filled aseptically with BHI broth (plus hemin plus vitamin K) to a volume of 100 ⁇ l by the use of a Beckman Biomek 1000 robotic instrument (Beckman Instruments, Palo Alto, CA) .
  • Peptides were tested in duplicate lanes by adding manually 100 ⁇ l of a 1.024 mg/ml peptide solution in water (w/v) to the top wells of a microtiter plate lane.
  • the peptide was diluted serially 1 : 2 by mixing and transferring 100 ⁇ l from the top well down to the bottom well in the lane by use of the Beckman Biomek 1000 (Beckman Instruments, Palo Alto, CA) . The last 100 ⁇ l from the bottom well was discarded. One hundred microtiters of the bacteria were added in BHI (plus hemin plus vitamin K x ) to each test well to give final peptide dilutions from 0.25 ⁇ l/ml. The plates were incubated in the anaerobic chamber at 37°C for 24-48 hours.
  • MIC minimum inhibitory concentration
  • CD-I male mice (average body weight, 22.8g) were inoculated with live E. coli strain 21915-1 (2.3 x 10 5 CFU mouse) by injection intraperitoneally.
  • Oct- (SEQ ID NO: 143) -NH 2 then was injected intravenously via the tail vein at 1 and 5 hours post-moculation.
  • Control mice were inoculated and treated with 0.9% saline. Each different treatment group had 10 mice per group. All control mice died.
  • Treatment doses of Oct- (SEQ ID NO: 143) -NH 2 were 1, 5, 10 and 20 mg/kg in toto, and resulted in 20%, 40%, 90% ana 90% survival at six days post-moculation, respectively.
  • Oct- (SEQ ID NO: 143) -NH was injected intravenously into male C57BL/6J mice (average body weight 20. Ig) approximately two minutes prior to mtrape ⁇ toneal injection of a solution of lipopolysaccharide (either 0.1 ⁇ g or 0.5 ⁇ g/mouse) from E . coll serotype C111:B4 and galactosamine (8 mg/mouse).
  • Treatmert doses of Oct-(SEQ ID NO: 143)-NH were 0, 5, 7.5, 10, 12.5 or 15 mg/kg (10 mice/group), and when administered prior to 0.5 ⁇ g lipopolysaccharide/mouse resulted in 10%, 0%, 30%, 0%, 50%, and 60% survival at five days post-lipopolysaccha ⁇ de administration, respectively.
  • the results were 40%, 90%, 100%, 100%, and 100% survival at five days post- lipopolysaccharide administration, respectively.
  • a stock solution (lOx) of 0.6 mM dye was prepared by adding 1.68 mg of (l-ethyl-2- (3- [1-ethylnaphthol (1, 2-d) -th ⁇ azolm-2- ylidene]-2-methylpropenyl) naphtho- (1,2-d) -thiazolium bromide (Signa E-7762) to 5 ml of 200 proof ethanol. 1 ml of this solution was added to 9 ml ethanol to give 0.06 mM of dye (60 ⁇ M dye) .
  • a stock solution of lipopolysaccha ⁇ de (LPS) from E . coli serotype 0111 :B4 was prepared at 1.5 mg/ml. 400 ⁇ l of this solution was mixed with 4.6 ml pyrogen free water to give a 120 ⁇ g/ml solution.
  • LPS lipopolysaccha ⁇ de
  • Row 1 and rows 3 through 12 of a microtiter plate were filled with 100 ⁇ l of pyrogen free water or with 10 mg/ml of bovme serum albumin. 200 ⁇ l of peptide then was added to row 2 of the microtiter plate at a concentration of 1 mg/ml. 200 ⁇ l of pyrogen free water was added to each of the control wells in two lanes (having dye and LPS but no peptide or having dye and no LPS and no peptide) . 100 ⁇ l then was serially diluted from row 2 through row 12 of the microtiter plate. 50 ⁇ l of PBS (pH 7.4) and 50 ⁇ l of the LPS solution then were added to row 1 of the plate (blank wells) .
  • the dye-buffer LPS mixture then was incubated for 10 minutes at room temperature in the dark.
  • 100 ⁇ l of the dye-LPS buffer mixture then was added to every well of the microtiter plate except to the blank wells and to the control lane that does not have LPS or peptide.
  • the plate was incubated for 10 minutes at room temperature in the dark, and the absorbance at 460 nm and 510 nm was read. From these absorbances, the LPS50 value, which is the concentration in mg/ml of peptide necessary to inhibit the binding of 50% of the lipopolysaccharide to the dye, was calculated.
  • the free base of an N-terminally modified peptide was generated by neutralizing the acetate or trifluoroacetate salt with saturated sodium carbonate solution.
  • the precipitated peptide was isolated either by centrifugation or by filtration, followed by washing with water.
  • a formaldehyde-sodium bisulfite complex was prepared by dissolving 5 gm of sodium bisulfite in a fixture of 75 ml of water and 4 ml of 35-40% formaldehyde with stirring. The solution was coole ⁇ and ethanol added. The precipitate obtained was filtered, washed with water, and dried. The product was recrystallized from water-ethanol .
  • the peptide free case was suspended in water and Formaldehyde-sodium bisulfite complex
  • the m vitro anti-microbial activities of the methane sulfonate derivatives were determined according to the methods described in Examples 1 and 2.
  • the activities against ATCC strains of S. aureus, E. coli , P. aeruginosa, and C. albicans, and the hemolysis of red blood cells were compared to the parent compound (Table IV) .
  • the activities for Compound 843 (Oct-SEQ ID NO:143) methane sulfonate against clinical strains of P. aeruginosa isolated from cystic fibrosis patients were compared to the parent compound (Table V) .
  • the activity for Compound 469 (Oct-SEQ ID NO: 27) methane sulfonate against P. gingival is was compared to the parent compound (Table VI) .
  • H sulfonate derivative C H OCT-SEQ ID NO: 27-NH 2 2 4 4-8 rn 64 100% m
  • H sulfonate derivative c SEQ ID NO: 154-NH 2 16 32 16-32 ND ND m ro SEQ ID NO: 154-OH 128 32 256 ND ND ⁇ > sodium methane sulfonate derivative
  • mice received single intravenous administrations of Compound 1324, the methane sulfonate analogue of Compound 843, and were monitored for survival for at least 4 days. There were no deaths at a dose of less than or equal to 180 mg/kg. This demonstrates that the chemical modification of Compound 843 to pro ⁇ uce Compound 1324 results in a 9-fold increase in safety.
  • Obiective :
  • mice were randomly assigned to one of ten groups (4 mice per group, except the highest dose group which only had one mouse) .
  • mice were administered intravenously 10 ml/kg of either 0
  • mice were monitored for survival for at least four days post-administration .
  • MTD maximum tolerated dose (non-lethal)
  • LD50 dose that was lethal to 50% of the animals
  • MTD i.v. 1180 mg/kg
  • LD50 i.v. between 200 and 282 mg/kg
  • Compound 843 (the parent compound of Compound 1324) has a significantly lower MTD of 20.0 mg/kg.
  • the chemical modification of Compound 843 yields Compound 1324 and produces a 9-fold increase in safety (see Fig. 1) .
  • This method was used to prepare the sodium methane sulfonate derivatives of SEQ ID NOS: 154-156.
  • the peptide salt (hydrochloride salt, acetate salt or trifluoroacetate salt) was taken into water and treated with an excess of 30% neutral formaldehyde solution (up to 12.5 equivalents for each am o group in the peptide) and an excess of IM sodium bicarbonate solution(up to 6.25 equivalents for each ammo group the peptide) .
  • the precipitated peptide adduct of formaldehyde was separated either by centrifugation or by filtration, washed with water and dried.
  • the formaldehyde adduct was suspended m water and an excess of sodium metabisulfite was added (up to 1.9 equivalents for each ammo group the peptide) to form the methane sulfornate derivative.
  • the clear solution was filtered through a 0.2 ⁇ m filter and lyophilyzed.
  • Human chronic myelogenous leukemia cells, K-562 (ATCC) were maintained in RPMI 1640 medium (GIBCO-BRL) supplemented with 10% fetal bovine serum.
  • Human melanoma cell line, WM 1617 (from the Wistar
  • Cytotoxicity assays were performed on the cell lines described above using CytoLite (Packard Instrument Company) following the manufacture's instruction. Briefly, 2xl0 4 cells/well were seeded into blac ⁇ , 96-well ViewPlates (Packard Instrument Company) in 100 ul/well growth medium. Serial dilutions of Magainin peptides were then added into the cells and tne plates incubated for 24 hours in a humidified 5% CO, atmosphere at 37°C. Following incubation, 25 ⁇ l/well of CytoLite activator solution was added followed by 125 ⁇ l/well of amplifier solution.
  • CytoLite Packard Instrument Company
  • Luminescence was measured on TopCount Microplate Scintillation and Luminescence counter (Packard Instrument Company) using a 1 second count time in SPC (single photon counting) mode at 25 °C . All assays were performed in triplicate. The peptides tested in this assay were MSI-78 (SEQ ID NO: 154-NH 2 ) , MSI-1858(SEQ ID NO: 154-NH 2 Methane
  • 2x10" cells/well are seeded into 96-well tissue culture plates in 100 ⁇ l/well growth medium and allowed to attach overnight in a humidified 5% C0 2 atmosphere at 37°C. Serial dilutions of Magainin peptides are then added into the cells and the plates are incubated for 18 hours. Following incubation, 0.2 ⁇ Ci/well of [ 3 H] -thymidine are added to the plates and further incubated for 6 hours at 37 °C . The cells are harvested, and the [ 3 H] -thymidine incorporation is determined by liquid scintillation counting. All assays are performed in triplicate.
  • MSI-344 SEQ ID NO: 154-OH
  • MSI-1857 SEQ ID NO: 154-OH methane sulfonate
  • a maximal tolerated dose (MTD) of a single intravenous (i.v.) administration or mtraperitoneal (l.p.) administration of MSI-344 or MSI-1857 ⁇ r 100 CD-1 ® BR mice were done.
  • the MTD of a single intravenous (_.v. a ⁇ mmistration was 10 mg/kg for MSI-344 and 60 mg/kg for MSI-1857.
  • the MTD of a single mtraperitoneal (l.p.) administration was less than 20 mg/kg for MSI-344 and between 50 and 100 mg/kg for MSI-1857.
  • the estimated LD50s (the dose that produces lethality in 50% of the animals injected) for intravenous administration were 12.5 and 73 mg/kg for MSI-344 and MSI- 1857, respectively.
  • the LD50s l.p. were 23.5 and 120 mg/kg for MSI-344 and MSI-1857, respectively.
  • MSI-344 A 5 mg/mL solution of MSI-344 was ma ⁇ e in saline (0.9% sodium chloride injection USP, Abbott, North Chicago, IL) corrected for peptide content. Solutions of MSI-344 at concentrations of 4, 3, 2, 1.5, 1, and 0.5 mg/mL were made by diluting the 5 mg/mL solution using saline as diluent. (Errors were made in solution preparation of the 4, 3, and 2 mg/mL solutions and results from groups with animals doses with these solutions, i.e. groups 12, 13, and 14, were eliminated from study analysis) . A 20 mg/mL solution of MSI-1857 was made in saline. Solutions of MSI-1857 at concentrations of 15, 10, 8, 6, and 5 mg/mL were made by diluting the 20 mg/mL solution using saline as diluent.
  • MSI-344 Another 5 mg/mL solution of MSI-344 was made in saline corrected for peptide content. Solutions of 4, 3 and 2 mg/mL were made by diluting the 5 mg/mL solution using saline as diluent. A 15 mg/mL solution of MSI-1857 in saline was made corrected for peptide content. A 10 mg/mL solution was made by diluting the 15 mg/mL solution with saline. Protocol :
  • mice (four mice per dose group) were intravenously or intraperitoneally administered a single bolus of MSI-344 or MSI-1857 using an injection volume of 10 mL/kg of the appropriate solution concentration. If the first two mice in a group died imme ⁇ iately (within 10 sees) after administration of a ⁇ ose then no other mice were injected with this dose of this compound (for ethical reasons). Survival was monitored daily for at least eight days post-admmistration. Study designs are presented in Tables IX and X. Toxicity Study # 262 was a supplement to Toxicity Study #261. Table IX. Study Design for Toxicity Study#261
  • mice Only two of four mice were m ⁇ ecte ⁇ because of resulting immediate ⁇ eath; for etmcai reasons the remaining two mice in this group were not injected.
  • the maximal tolerated doses (MTDs) for intravenous administration of MSI-344 and MSI-1857 were 10 and 60 mg/kg, respectively, in mice.
  • the MTDs for intraperitoneal administration of MSI-344 and MSI- 1857 were ⁇ 20 and 50 mg/kg, respectively.
  • the approximate dose that caused lethality in 50% of dosed animals (LD50) for intravenous administration of MSI-344 and MSI-1857 in mice were 12.5 and 73 mg/kg, respectively.
  • the LD50s for intraperitoneal administration of MSI-344 and MSI- 1857 were approximately 23.5 and 120 mg/kg, respectively.
  • MSI-344 SEQ ID NO : 154-OH
  • MSI-344 or MSI- 1857 were injected with various doses of MSI-344 or MSI-1857 twice or thrice per week. Survival and body weight were monitored. On a twice per week dosing schedule, the MTDs of MSI-344 and MSI-1857 were 12 and 134 mg/kg/week, respectively. This demonstrated that MSI-1857 had an 11-fold increase m safety index compared to MSI-344.
  • SCID mice To determine estimates of MTDs of MSI-344 and MSI-1857 when administered intraperitoneally on repeat-dose regimens in SCID mice. The selection of SCID mice was based on the future plans of using this strain as host for xenograft human tumor efficacy models, and a pre-requisite for determining MTD for chronic administration that may be used during the efficacy model .
  • SCID mice Two groups of sixty SCID male mice (Fox Chase C.B- 17/IcrTac-scidfDF, Taconic Labs) exhibited a body weight range of 19.6-25.8 gms at study initiation. SCID mice are immunocompromised and therefore they were housed and handled in sterile environments. They had access to sterile water and sterilized chow ad lib.
  • MSI-344 A 1 mg/mL solution of MSI-344 was made in saline (0.9% sodium chloride injection USP, Abbott, North Chicago, IL) corrected for peptide content. Solutions of MSI-344 at concentrations of 0.6, 0.3, and 0.1 mg/mL were made by diluting the 1 mg/mL solution using saline as diluent. A 10 mg/mL solution of MSI-1857 was made in saline .
  • Solutions of MSI-1857 at concentrations of 6.7, 3.3, 3, 2, and 1 mg/mL were made by diluting the 10 mg/mL solution using saline as diluent.
  • Protocol
  • mice four mice per dose group were intraperitoneally administered MSI-344 or MSI-1857 using an injection volume of 10 mL/kg of the appropriate solution concentration on a twice per week (Groups 1 through 7) or thrice per week (Groups 8 through 15) dosing schedule every week for 20 weeks. Survival was monitored daily and body weights were recorded three times per week. An interim analysis was performed at 3 weeks and the results of this interim analysis is presented in the present report . Study design is presented in Table XII.
  • Sodium metabisulfite was dosed at 135 mg/kg/week on three injection per week schedule. This dose is equidose to the quantity of sodium metabisulfite that was used to prepare the MSI-1857 in the MSI-1857 group dosed at 90 mg/kg/week. There was no lethality in this group nor was there any effect of sodium metabisulfite on body weight.
  • the maximal tolerated dose (MTD) of MSI-1857 (134 mg/kg/week) was 11-fold greater than MSI-344 (12 mg/kg/week) in SCID mice.
  • MTD maximal tolerated dose
  • MSI-1857 was greater than or equal to 90 mg/kg/week and the MTD of MSI-344 was greater than or equal to 18 mg/kg/week.
  • Sodium metabisulfite was administered at a dose that was equivalent to the amount of metabisulfite contained in the MSI-1857 group dosed at 90 mg/kg/week (the dose had been corrected for peptide content) . Any toxicity or changes observed in the MSI-1857 group could have been due to the peptide dose or metabisulfite dose, if there was any free, unbound metabisulfite in the solution. The lack of any effect of the metabisulfite on lethality or body weight would suggest that the transient weight loss seen in the MSI-1857 group was not due to free metabisulfite. In addition, even complete release of bound metabisulfite from the high dose group would not cause any deleterious effects on survival or body weight.
  • mice Thirty-six mice were intravenously administered a single bolus of MSI-1324 at a dose of 140 mg/kg corrected for peptide content using an injection volume of 10 mL/kg of the 14 mg/mL solution.
  • Four mice were not injected and supplied pre-dosing blood samples. Terminal blood samples were obtained via cardiac puncture in halothane- anesthetized mice at timepoints of 0 (prior to dosing), 5, 15, 30, 60 min, 2, 4, 6, 24, and 48 hours post-dose. Two to four mice were terminally bled at each timepoint. The blood samples were centrifuged and plasma transferred into separate tubes for bioassay analysis. The bioassay used was antibacterial zone clearing (disk diffusion) on Escherichia coli lawns. Results :
  • Figure 15 shows the standard curve of E. coli zone clearing of MSI-1324 used in the bioassay.
  • Figure 16 shows the plasma concentrations (estimated from the bioassay) in mice at various times after a dose of 140 mg/kg. Peak concentrations of approximately 600 ug/mL were observed at 5 min post-dose (the first sampling timepoint post-dose) . Concentrations were below the limit of detection (approximately 150 ug/mL) of the bioassay at 60 min post-dose. The insert in the figure shows no detectable level at later time points.
  • MSI-1324 showed peak concentrations at 5 min after intravenous administration of 140 mg/kg in mice. Plasma levels were below detection at 60 min and later timepoints.
  • mice Female nude mice weighing approximately 20g are implanted s.c. by trocar with fragments of SK-MES human lung tumors harvested from s.c. growing tumors in nude mice hosts, while for the human breast tumor model, the mice are injected in the mammary fat pad (mfp) with 1EE6 MDA-MB-435 cells from culture.
  • mfp mammary fat pad
  • tumors reach approximately 5mm x 5mm (about ten to fourteen days after inoculation)
  • the animals are pair-matched into treatment and control groups. Each group contains 10 tumored mice, each of which is ear-tagged and followed individually throughout the experiment. The administration of drugs or vehicle begins the day the animals are pair-matched (Day 1) .
  • mice are dosed intraparationally either twice or three times a week. Mice are weighed twice weekly, and tumor measurements are taken by calipers twice weekly, starting on Day 1. These tumor measurements are converted to mg tumor weight by a well-known formula, L5 x /2. The experiment is terminated when control tumors reach a size of 1 gram for the breast tumor model and a size of 2 grams for the lung tumor. Upon termination, all mice are weighed, sacrificed, and their tumors excised. Tumors are weighed, and the mean tumor weight per group is calculated. In this model, the mean treated tumor weight / mean control tumor weight x 100 (T/C) is subtracted from 100% to give the tumor growth inhibition (TGI) for each group.
  • TGI tumor growth inhibition
  • mice with partial or total tumor regressions can be kept alive past the termination date to see whether they live to become long term, tumor-free survivors.
  • peptides or proteins of the present invention 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.
  • peptides or proteins and/or agent as hereinabove described may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful microorganisms including protozoa, viruses, parasites, fungi, and the like.
  • the peptides or proteins may be administered to a host, in particular an animal, in an effective antibiotic and/or anti-tumor and/or anti-viral and/or anti-microbial and/or spermicidal and/or anti-fungal and/or anti-parasitic amount, or in an amount effective to stimulate wound healing in a host, or in an amount effective in treating sepsis or septic shock in a host.
  • the peptides or proteins may be administered either alone or in combination with an ion having pharmacological properties, an antibiotic, or an ion channel forming peptide or protein as hereinabove described. When the peptide or protein is administered in combination with an ion having pharmacological properties, the activity of the peptide or protein is potentiated.
  • the peptide or protein is administered in combination with an agent as hereinabove described, it is possible to administer the peptide and agent in separate forms.
  • the agent may be administered systemically and the peptide or protein may be administered topically.
  • the peptide or protein When the peptide or protein is administered topically, it may be administered in combination with a water-soluble vehicle, the water-soluble vehicle being in the form of an ointment, cream, lotion, paste, or the like.
  • water-soluble vehicles which may be employed include, but are not limited to, glycols, such as polyethylene glycol, hydroxycellulose, and KY Jelly.
  • the water-soluble vehicle is preferably free of an oily substance.
  • the peptide or protein may also be employed alone, or in combination with an ion having pharmacological properties, as hereinabove described, in the form of an oral composition for oral hygiene.
  • compositions and materials used for oral hygiene purposes include, but are not limited to, toothpastes, mouthwashes, tooth gels, and tooth powders.
  • Such composition may thus be used to treat or prevent periodontal diseases, to prevent or reduce plaque, gingivitis, and/or to prevent or treat or reduce dental caries.
  • the peptide and ion having pharmacological properties may be used to inhibit, prevent, or destroy the growth of Streptococcus mutans, which is associated with dental caries and periodontal disease.

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Abstract

L'invention concerne des peptides biologiquement actifs à toxicité réduite et des procédés relatifs à leur élaboration. Lesdits peptides, pouvant être non substitués ou à substitution N-terminale, sont représentés par la formule (I) . Dans ladite formule, X est un peptide ou une protéine à formation de canal ionique de type amphiphile, T est une fraction lipophile ou hydrogène, et W est T ou hydrogène. De préférence, T est représenté par la formule (II). Dans ladite formule, R est hydrocarbure (alkyle ou aromatique ou alkylaromatique) ayant au moins 2 et au maximum 10 atomes de carbone. T est de préférence un groupe octanoyle. Les peptides et les protéines considérés ont une activité biologique antimicrobienne et antitumorale améliorée ainsi qu'une toxicité réduite. Le procédé préféré de réduction de la toxicité consiste à élaborer des dérivés apparentés de méthane sulfonate ou des analogues. Par ailleurs, les composés décrits peuvent être utilisés en traitement de maladie infectieuse, de choc septique, et d'infection pulmonaire, comme par exemple dans le cas de la mucoviscidose.
PCT/US1998/014610 1997-07-15 1998-07-15 Peptides biologiquement actifs a toxicite reduite chez l'animal et procedes d'elaboration WO1999003488A2 (fr)

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EP98933343A EP1001800A2 (fr) 1997-07-15 1998-07-15 Peptides biologiquement actifs a toxicite reduite chez l'animal et procedes d'elaboration
AU83005/98A AU8300598A (en) 1997-07-15 1998-07-15 Biologically active peptides with reduced toxicity in animals and a method for preparing same
JP2000502785A JP2001510164A (ja) 1997-07-15 1998-07-15 動物に対する毒性を減じた生物活性ペプチドおよび同ペプチドを調製する方法。
CA002294518A CA2294518A1 (fr) 1997-07-15 1998-07-15 Peptides biologiquement actifs a toxicite reduite chez l'animal et procedes d'elaboration

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8318899B2 (en) 2008-01-24 2012-11-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Lytic domain fusion constructs and methods of making and using same
US8609608B2 (en) 2007-01-16 2013-12-17 C3 Jian, Inc. Antimicrobial peptides
US20140051628A1 (en) * 2007-08-03 2014-02-20 PharmalN Corporation Composition for long-acting peptide analogs
US9492563B2 (en) 2012-10-30 2016-11-15 Esperance Pharmaceuticals, Inc. Antibody/drug conjugates and methods of use

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024138A1 (fr) * 1992-06-01 1993-12-09 Magainin Pharmaceuticals, Inc. Peptides biologiquement actifs presentant des substitutions n-terminales
WO1995019370A1 (fr) * 1994-01-18 1995-07-20 Magainin Pharmaceuticals Inc. Peptides amphiphiliques formant des canaux ioniques et presentant des modifications n-terminales

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024138A1 (fr) * 1992-06-01 1993-12-09 Magainin Pharmaceuticals, Inc. Peptides biologiquement actifs presentant des substitutions n-terminales
WO1995019370A1 (fr) * 1994-01-18 1995-07-20 Magainin Pharmaceuticals Inc. Peptides amphiphiliques formant des canaux ioniques et presentant des modifications n-terminales

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8609608B2 (en) 2007-01-16 2013-12-17 C3 Jian, Inc. Antimicrobial peptides
AU2008251748B2 (en) * 2007-01-16 2014-02-27 C3 Jian, Inc. Novel antimicrobial peptides
US20140051628A1 (en) * 2007-08-03 2014-02-20 PharmalN Corporation Composition for long-acting peptide analogs
US9090664B2 (en) * 2007-08-03 2015-07-28 Pharmain Corporation Composition for long-acting peptide analogs
US9657078B2 (en) 2007-08-03 2017-05-23 Pharmain Corporation Composition for long-acting peptide analogs
US8318899B2 (en) 2008-01-24 2012-11-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Lytic domain fusion constructs and methods of making and using same
US8546535B2 (en) 2008-01-24 2013-10-01 Esperance Pharmaceuticals, Inc. Lytic domain fusion constructs and methods of making and using same
US9255134B2 (en) 2008-01-24 2016-02-09 Esperance Pharmaceuticals, Inc. Lytic domain fusion constructs and methods of making and using same
US9492563B2 (en) 2012-10-30 2016-11-15 Esperance Pharmaceuticals, Inc. Antibody/drug conjugates and methods of use
US10233214B2 (en) 2012-10-30 2019-03-19 Esperance Pharmaceuticals, Inc. Antibody/drug conjugates and methods of use

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