HK1187357A - Antimicrobial peptides comprising an arginine- and/or lysine-containing motif - Google Patents

Abstract

The present invention relates to a peptide comprising amino acids according to Formula (I): ((X)1(Y)m)n wherein 1, m and n are integers from 0 to 10; X and Y, which may be the same or different, are an amino acid selected from the group consisting of hydrophobic amino acids and/or cationic amino acids, for use as a medicaments.

Description

Antimicrobial peptides comprising arginine-and/or lysine-containing motifs

The present invention provides antimicrobial peptides. The invention also relates to pharmaceutical compositions comprising the antimicrobial peptides and to the use of the peptides in the treatment of, inter alia, microbial infections.

An important family of endogenous antimicrobial peptides, the β -defensins, are secreted by epithelial cells lining the digestive, respiratory and genitourinary tracts of higher mammals. Keratinocytes in the skin also produce the beta-defensin. Their main role is to provide the first important line of defense against infections caused by pathogenic microorganisms through the above routes.

Defensins are the most studied class of antimicrobial peptides. This class of substances consists of cysteine-rich molecules with three disulfide bridges. It is present in plants, insects and various mammals. There are two classes of defensins in humans, which differ in the spacing and bond between the 6 cysteine residues. The first of these two classes of defensins is the alpha-defensin (type 6), which is isolated from the neutrophil granulocytes (HNP1-4, human neutrophilic peptide) and is located in the Paneth cells of the gastrointestinal tract (alpha-defensins 5 and 6). The second class, β -defensins, are longer, more basic, and are expressed in the mucosa in epithelial and keratinocytes cells lining and/or contained and/or present in the digestive, respiratory and genitourinary tracts and skin. hBD1 (human β -defensin 1) is constitutively secreted and human β -defensins 2, 3 and 4(hBD2, hBD3 and hBD4) are produced in response to infection or inflammation. Stimulation of bacteria, particularly flagellates (Harder et al, Nature1997;387:861) and IL1[ alpha ] and IL1[ beta ] (interleukin 1) (Liu et al, J Invest Dermatol2002;118; 275-. Tumor necrosis factor alpha (TNF-alpha) and Lipopolysaccharide (LPS) may also act to induce the expression of hBD2 at certain tissue sites. In vitro experiments have revealed that hBD2 has activity against gram-negative bacteria such as escherichia coli (e.coli) and to a lesser extent against gram-positive bacteria such as streptococcus pneumoniae (str. hBD2 also showed killing activity against the yeast Candida albicans (yeast Candida albicans) in vitro. Bacterial stimulation, TNF- α and especially interferon γ (IFN γ) induce the expression and secretion of hBD3, these factors having the common property of being molecules involved in inflammatory processes.

In addition to providing potent, constitutive, and regulated broad spectrum intrinsic antimicrobial protection, these molecules, in particular hBD2, also have the ability to mobilize the adaptive arm of the immune response (adaptive arm) through chemotactic effects on immature dendritic cells and memory T lymphocytes (Yang et al, Science1999;286: 525-.

Importantly, there is evidence that β -defensins not only provide protection against infections caused by pathogenic microorganisms, but also play a key role in regulating and maintaining the optimal density and diversity of the symbiotic microbial ecosystem essential to the body, such as on the skin, in the gastrointestinal and reproductive tracts (Ganz, T, Nat Rev immunol.20033(9): 710-20).

The mode of action of the beta-defensin is such that it is substantially non-toxic to the host cell at active concentrations. Thus, β -defensins are a potential target for the treatment of a wide range of infections. However, the production of natural forms of defensins in recombinant systems is technically challenging, resulting in low yields. Furthermore, there is increasing evidence that beta-defensins are potent inflammatory compounds through their chemotactic effects (Yang et al, Science1999;286: 525-; Van Wettinget al Inflamm Res.2002;51(1):8-15; Niyonsaba et al curr drags TargetsInflamm Allergy2003;2(3): 224-) -231). Together, these factors make natural defensins unsuitable for therapeutic applications.

Beta-defensins are highly salt sensitive (Porter et al, Infect. Immun.1997;65(6):2396-401; Bals et al J Clin invest.1998;102(5):874-80; Valore et al, JClin Invest1998;101(8):1633-42; Goldmann et al, Cell1997;88(4):553-609; Singh et al, Proc Natl Acad Sci USA95(25): 14961-6). For this reason, beta-defensins fail to provide antimicrobial protection under conditions such as cystic fibrosis, where the respiratory epithelium produces large amounts of beta-defensin in response to persistent bacterial infection associated with the disease state, but due to the disease stateImbalance of ion transport across respiratory epithelial membranes, inactivation of beta-defensins, which imbalance leads to increased cation reabsorption (especially Na)⁺) And increased chloride secretion Donaldson SH and Boucher RC. curr. opin. puim. Med.2003Nov;9(6):486-91; Davies JC. Pediatr Pulmonol suppl.2004;26: 147-8).

Thus, there is a need for additional agents that can be used to treat microbial infections.

The present invention identifies peptides that unexpectedly have improved antimicrobial activity compared to natural defensins.

According to a first aspect of the present invention there is provided a peptide comprising 3 to 200D and/or L amino acids, which may be the same or different, wherein the amino acids are selected from hydrophobic amino acids and/or cationic amino acids. The peptide may comprise 3 to about 100D and/or L amino acids, for example 3 to 50D and/or L amino acids, including 4 to about 50D and/or L amino acids.

The peptides of the invention are particularly useful for the treatment or prevention of microbial infections.

In another aspect of the invention, there is provided a peptide comprising amino acids according to formula I:

((X)_l(Y)_m)_n (I)

wherein l and m are integers from 0 to 10, for example from 0 to 5; n is an integer from 1 to 10; x and Y are amino acids selected from hydrophobic amino acids and/or cationic amino acids, said X and Y may be the same or different.

In a preferred aspect of the invention, the peptide has from 3 to 200 amino acids, for example from 3, 4, 5,6 or 7 to 100 amino acids, including from 3, 4, 5,6 or 7 to 20, 25, 30, 35, 40 or 42 amino acids.

The peptides of the invention may comprise 100 to 200 amino acids, 27 to 100 amino acids, 28 to 86 amino acids, 7 to 27 amino acids, or 3 to 14 amino acids.

Preferably, the peptide comprises 3 to 15 amino acids, for example 3 to 7 amino acids.

In another preferred aspect, the peptide further comprises one or more cysteine residues, for example comprising up to 6 cysteine residues, for example comprising 1, 2, 3, 4, 5,6 cysteine residues.

In a preferred aspect of the invention, there is provided a peptide comprising amino acids represented by the general formula II:

C((X)_l(Y)_m)_nC((X)_l(Y)_m)_n (II)

wherein C is cysteine, l, n and m are integers from 0 to 10, X and Y are amino acids selected from hydrophobic amino acids and/or cationic amino acids, said X and Y may be the same or different.

In another preferred aspect of the invention, there is provided a peptide comprising amino acids represented by the general formula III:

C((X)_l(Y)_m)_nC((X)_l(Y)_m)_nC((X)_l(Y)_m)_nC((X)_l(Y)_m)_nC (III)

wherein C, X, Y, l, m and n are as defined herein.

In another preferred aspect of the present invention, there is provided a peptide comprising amino acids represented by the general formula IV:

C((X)_l(Y)_m)_nC((X)_l(Y)_m)_nC((X)_l(Y)_m)_nC((X)_l(Y)_m)_nCC (IV)

wherein C, X, Y, l, m and n are as defined herein.

Since the peptide of the present invention is structurally simpler than the natural β -defensin, the peptide of the present invention is easy to produce and has a high yield. Moreover, the peptides are substantially salt insensitive and non-hepatotoxic. Furthermore, their physical form, rather than their biological metabolic (i.e., direct membrane disruption, relatively targets a constituent element of an important biological metabolic pathway), mode of action, if not excluded, also minimizes the likelihood that the target microorganism may develop resistance to the antimicrobial agent.

It is known to those skilled in the art that amino acids can be classified into different types mainly according to the chemical and physical properties of the side chains of the amino acids. For example, certain amino acids are generally considered hydrophilic or polar amino acids while other amino acids are generally considered hydrophobic or non-polar amino acids. The terms "hydrophobic" and "cationic" as used herein may refer to amino acids having a hydrophobicity of-1.10 or greater and/or a net charge of 0 or greater as described in Fauchere and Pluska Eur.J.Med chem.10:39 (1983). Hydrophobic or non-polar amino acids may also refer to amino acids having side chains that are uncharged at physiological pH, which are non-polar and generally immiscible with aqueous solutions.

In a preferred aspect of the invention, X and/or Y are selected from hydrophobic amino acids consisting of glycine, leucine, phenylalanine, proline, alanine, tryptophan, valine, isoleucine, methionine, tyrosine and threonine and/or cationic amino acids consisting of ornithine, histidine, arginine and lysine. X and/or Y may be a D or L-amino acid. Furthermore, X and/or Y may be amino acids used interchangeably.

The invention also includes known isomers (structural, stereo, conformational and configurational) and structural analogs of the above amino acids, as well as analogs that are naturally occurring (e.g., post-translational modifications) or chemically modified including, but not limited to, phosphorylation, glycosylation, sulfonylation and/or hydroxylation.

Typically, the peptides of the invention do not comprise amino acids such as aspartic acid, glutamic acid, asparagine, glutamine or serine, but even if these amino acids are present, certain peptides of the invention may have activity.

The peptides of the invention may comprise one or more additional amino acid residues adjacent to one or both terminal cysteine residues of formula II, III or IV, e.g. the peptides may contain up to 10 (e.g. 1, 2, 3, 4, 5,6, 7, 8, 9 or 10) additional amino acid residues. Preferably, the additional amino acid residue is a non-cysteine residue. More preferably, the additional amino acid is X and/or Y.

Furthermore, the amino acid sequence of the peptide may be modified so as to obtain a peptide variant comprising at least one amino acid residue of the peptide substituted for another amino acid residue, said substitution comprising the use of D-form for L-form.

One or more residues of a peptide may be exchanged with another to alter, enhance or protect the biological activity of the peptide. For example, such variants can have at least about 10% of the biological activity of the corresponding non-variant peptide. Conservative amino acids are often used, i.e., substitutions with amino acids having similar chemical and physical properties as described above.

Thus, for example, a conservative amino acid substitution may involve the substitution of lysine for arginine, ornithine or histidine; one hydrophobic amino acid is replaced by another. After introduction of the substitutions, the variants are screened for biological activity.

The peptide may contain at least 4 amino acids, for example 4 to 50 amino acids, or 4 to 50 amino acids such as 20 to 45 amino acids, for example 20, 25, 30, 35, 40, 42 or 45 amino acids.

In a preferred aspect of the invention, X and Y are the same and are leucine or glycine.

In another preferred aspect of the invention, X is leucine and Y is glycine.

In another preferred aspect, X and Y are the same and are lysine or arginine. The present invention therefore provides a peptide selected from the group consisting of poly-L-lysine, poly-D-lysine, poly-L-arginine and poly-D-arginine.

In another preferred aspect, X is lysine and Y is arginine.

In the peptide of the present invention, l and m may be 0,1, 2, 3, 4, 5,6, 7, 8, 9 or 10 and n may be 1, 2, 3, 4, 5,6, 7, 8, 9 or 10.

In the peptides of the invention, l may be 1, n may be 1 and m may be 4 to 9, for example m may be 3, 4, 5,6, 7, 8 or 9.

In the peptides of the invention, l, n and/or m may be 1 to 5, for example 1, 2, 3, 4 or 5.

Preferred peptides are acyclic. The peptides may be linear, i.e. linear or branched.

The term "peptide" as used herein refers to a large number of amino acid residues joined, typically by peptide bonds. It can be used interchangeably with and has the same meaning as polypeptides and proteins.

In one embodiment of the invention, the peptide comprises an amino acid sequence selected from the group consisting of:

CGGGGGGCGGGGCGGGGGGGGGCGGGGGGCC (i)

CLLLLLLCLLLLCLLLLLLLLLCLLLLLLCC (ii)

CLGLGLGCGLGLCLGLGLGLGLCGLGLGLCC (iii)

CRKRKRRCRKRKCKRKRKEKRKCRKRKRKCC (iv)

KKK (v)

KKKKKKK (vi)

RRR (vii)

RRRRRRR (viii)

in another aspect of the invention, the peptide comprises at least one of the amino acid sequences (i) to (viii) and additional amino acid residues adjacent to one or both terminal cysteine residues. Thus, in another embodiment of the invention, there is provided a peptide comprising an amino acid sequence selected from the amino acid sequences shown in figure 1.

The peptides of the invention are typically synthetic peptides. The peptides may be isolated, purified peptides or variants thereof, which may be synthesized in vitro, for example by solid phase peptide synthesis, enzyme catalyzed peptide synthesis or using recombinant DNA techniques.

To identify active peptides that have little or no undesirable toxicity to mammalian cells, individual peptides or peptide libraries can be prepared and the individual peptides or peptides from the peptide libraries can be screened for antimicrobial activity and toxicity, including but not limited to antifungal, antibacterial, antiviral, antiprotozoal, antiparasitic activity and toxicity.

The peptides of the invention may exist in different forms, such as free acids, free bases, esters or other prodrugs, salts and tautomers, and the invention includes all variant forms of the compounds.

Thus, the invention includes salts or prodrugs of the peptides or peptide variants of the invention.

The peptides of the invention may be administered in the form of pharmaceutically acceptable salts. The pharmaceutically acceptable salts of the invention can be synthesized from the parent peptide, which contains a basic or acidic moiety, by conventional chemical methods. Typically, the salts are prepared by reacting these peptides in free acid or free base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two, non-aqueous solvents such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile being generally preferred. Suitable salts are given in Remington's pharmaceutical Sciences, 17th ed., Mack publishing company, Easton, Pa., US,1985, p.1418, which is incorporated herein by reference; see also Stahl et al, Eds, "Handbook of pharmaceutical salts Properties Selection and Use" (Handbook of drug salt Properties), Verlag Helvetica Chimica Acta and Wiley-VCH, 2002.

The invention therefore includes pharmaceutically acceptable salts of the disclosed peptides wherein the parent compound is modified by forming an acidic or basic salt thereof, for example a conventional non-toxic salt or a quaternary ammonium salt formed, for example, from an inorganic or organic acid or base. Examples of acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmitate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Basic salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine and N-methyl-D-glutamine, and salts with amino acids such as arginine and lysine. Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, dodecyl, tetradecyl and octadecyl chlorides, bromides and iodides; such as benzyl and phenethyl bromides and the like.

The carboxyl salts of the peptides or peptide variants of the invention may be prepared using conventional methods by contacting the peptide with one or more equivalents of the desired base, for example a metal hydroxide base such as sodium hydroxide; metal carbonates or bicarbonates such as sodium carbonate or sodium bicarbonate; or amines such as triethylamine, triethanolamine, etc.

The N-acyl derivative of the amino group of the peptide or peptide variant of the present invention can be prepared by using an N-acyl protected amino acid for final concentration, or by acylating a protected or unprotected amino acid. The O-acyl derivatives can be prepared, for example, by acylating a peptide or peptide resin having a free hydroxyl group. Any of the above acylations can be carried out using standard acylating agents such as acyl halides, anhydrides, acyl imidazoles, and the like.

The invention includes prodrugs of active drug classes of the peptides, for example where one or more functional groups that are protected or derivatised modified may be converted in vivo to the functional group, for example where carboxylic esters may be converted in vivo to the free acid, or where protected amines may be converted to the free amino group. The term "prodrug" as used herein denotes in particular a structure which can be rapidly converted in vivo to the parent structure, for example by hydrolysis in blood. T.Higuchi and V.Stella, Pro-drugs as Novel Delivery Systems, Vol.14of the A.C.S.Sympossium Series, Edward B.Roche, ed., Bioreproducible Carriers in Drug Design, American pharmaceutical Association and Pergamon Press,1987, H Bundgaard, ed, Design of Prodrugs, Elsevier,1985, and Judkins, et al synthetic Communications,26(23),4351-4367(1996) provide detailed discussion, which is incorporated herein by reference.

Thus, prodrugs include drugs having functional groups that have been converted to their reversible derivatives. Typically, these prodrugs are converted to the active drug by hydrolysis. Examples that may be mentioned are the following:

prodrugs also include compounds that can be converted to the active drug by oxidation or reduction. Examples that may be mentioned are the following:

oxidative activation

N-and O-dealkylation

Oxidative deamination

N-oxidation

Epoxidation of

Reduction activation

Reduction of azo

Reduction of sulfoxide

Disulfide reduction

Bioreduction alkylation

Reduction of nitro groups

It should also be mentioned that metabolic activation of the prodrug is nucleotide activation, phosphorylation activation and decarboxylation activation.

The use of protecting Groups is fully described in "Protective Groups in Organic Chemistry", edited by J W F McOmie, Plenum Press (1973) and "Protective Groups in Organic Synthesis", 2nd edition, T W Greene & PG M Wutz, Wiley-Interscience (1991).

Thus, it will be appreciated by those skilled in the art that although protected derivatives of the peptides may not possess pharmacological activity per se, they may be administered, for example parenterally or orally, and thereafter metabolised in the body to form pharmaceutically active compounds. Thus, such derivatives are examples of "prodrugs". All prodrugs of the compounds are included within the scope of the invention.

Another aspect of the invention provides a pharmaceutical composition comprising a pharmaceutically effective dose of at least one peptide of the invention, or two or more peptides of the invention.

These peptides may also include pharmaceutically acceptable carriers, excipients or diluents. The term "pharmaceutically acceptable" as used herein, means a compound, material, composition, and/or dosage form, which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or, in some cases, animals, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The peptides of the invention are particularly useful as antimicrobial peptides, for example against bacteria, fungi, yeast, parasites, protozoa and viruses. The term "antimicrobial peptide" as used herein is defined as any peptide having an activity that kills microorganisms and/or inhibits only their growth (microbiostatic) and non-exclusively includes any peptide that is said to have antibacterial, antifungal, antimycotic, antiparasitic, antiprotozoal, antiviral, antiinfective and/or bactericidal, algicidal, amoebic, microbiocidal, bactericidal, fungicidal, parasiticidal, protozoal (protozoacidal) properties.

Accordingly, the present invention also provides a peptide of the invention for use as a medicament. The peptides of the invention can be used as antimicrobial agents for in vivo and in vitro treatments for in vivo reinfusion (ex vivo).

In a preferred aspect, the invention provides the use of a peptide of the invention in the manufacture of a medicament for the treatment of a microbial infection.

"microbial infection" refers to an infection caused by a bacterial, parasitic, protozoal, viral, or fungal pathogen. A "pathogen" is generally defined as any organism that causes disease.

The bacterial pathogen may be from a bacterial species selected from the group consisting of: staphylococci (Staphylococcus spp.), such as Staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis (Staphylococcus epidermidis); enterococcus (enterococcsp), such as Enterococcus faecalis (Enterococcus faecalis); streptococcus pyogenes (streptococcus pyogenes); listeria (Listeria spp.); pseudomonas spp (Pseudomonas spp.); mycobacteria (Mycobacterium spp.) such as Mycobacterium tuberculosis (Mycobacterium tuberculosis); enterobacter (Enterobacter spp.); campylobacter (Campylobacter pp.); salmonella (Salmonella spp.); streptococcus (Streptococcus spp.) such as Streptococcus Group A or B (Streptococcus Group A or B), Streptococcus pneumoniae (Streptococcus pneumoniae); helicobacter sp, such as Helicobacter pylori (Helicobacter pylori); neisseria species (Neisseria spp.), such as Neisseria gonorrhoeae (Neisseria gonorrhea), Neisseria meningitidis (Neisseria meningitidis); borrelia burgdorferi (Borrelia burgdorferi); shigella (Shigella spp.), for example Shigella flexneri; escherichia coli (Escherichia coli); haemophilus (Haemophilus spp.) such as Haemophilus influenzae (Haemophilus influenzae); chlamydia species (Chlamydia spp.) such as Chlamydia trachomatis (Chlamydia trachomatis), Chlamydia pneumoniae (Chlamydia pneumoniae), Chlamydia psittaci (Chlamydia psittaci); francisella tularensis (Francisella tularensis); bacillus sp, Bacillus anthracis; clostridia (Clostridium spp.) such as Clostridium botulinum (Clostridium botulinum); yersinia spp, such as Yersinia pestis (Yersinia pestis); treponema spp; burkholderia spp, for example Burkholderia rhinoceros (Burkholderia mallei) and Burkholderia pseudorhinoceros (B pseudomallei).

The viral pathogen may be from a virus selected from the group consisting of: human Immunodeficiency Virus (HIV1&2) (Human Immunodeficiency Virus (HIV1& 2)); human T Cell leukemia Virus (HTLV1&2) (Human T Cell Leukaemia Virus (HTLV1& 2)); ebola virus (Ebola virus); human papilloma virus (human papillomavir) (e.g., HPV-2, HPV-5, HPV-8HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54 and HPV-56); papovavirus (papovavirus); rhinovirus (rhinovirus); poliovirus (poliovirus); herpes virus (herpesvirus); adenovirus (adenoviruses); epstein Barr virus; influenza virus (inflenzavirus); hepatitis B and C viruses (hepatis B and C viruses); smallpox virus (Variola virus); rotavirus (rotavirus) or SARS coronavirus (SARScoronavirus).

The parasitic pathogen may be from a parasitic pathogen selected from the group consisting of: trypanosomes (Trypanosoma spp.) (Trypanosoma cruzi), Trypanosoma brucei (Trypanosoma brucei), Leishmania (Leishmania spp.), Giardia (Giardia spp.), Trichomonas (Trichomonas spp.), amebiasis (entamoebasp.), genisis (Naegleria spp.), echinospora (Acanthamoeba spp.), hematophagous (Schistosoma spp.), Plasmodium (Plasmodium spp.), cryptosporidium (cryptosporidium spp.), microsporum (cryptosporium spp.), microsporidium, microsporidia (Isospora spp.), Trypanosoma (Plasmodium spp.), Trypanosoma pard (Plasmodium spp.), Trichomonas (Trichomonas spp.), and Trichomonas (Trichomonas).

The fungal pathogen may be from a fungal pathogen selected from the group consisting of: candida (Candida spp.) (e.g. Candida albicans), Epidermophyton (epimorphon spp.), ectochytrium (Exophiala spp.), Microsporum (Microsporum spp.), Trichophyton (Trichophyton spp.) (e.g. Trichophyton rubrum (t. rubrum) and Trichophyton interdigitatum), Trichophyton (tineum (tinctorium), Aspergillus (Aspergillus spp.), blastomyceliophyton (Trichophyton spp.), coccidiodes (coccospora spp.), Trichophyton (Trichophyton spp.), Candida spp.), trichophyceae (Trichophyton. sp.), trichophyceae (trichophytin), trichophytin (trichophytin spp.), trichophytin (trichophytin), trichophytin (trichophy, Acremonium sp, Actinomyces sp, Malaysia sp, Chrysosporium sp, Glonospora sp, Beauveria sp, Chrysosporium sp, Conidiobolus sp, Cunninghamella sp, Fusarium sp, Geotrichum sp, Cladosporium sp, Phycomyces sp, Nosporum, Phycomyces sp, Nocardia sp, rhizopus (Pyrenochaeta spp.), Rhizomucor (Rhizomucor spp.), rhizopus (Rhizopussp.), Rhizopus (Rhizopuspp.), Rhodotorula (Rhodotorula spp.), Saccharomyces (Saccharomyces spp.), foot-actinomycete (Scedosporium spp.), Scedosporium (Scedosporium spp.), Sporobolomyces (Sporobolomyces spp.), Syncephalopsis (Syncephalagrum spp.), Trichoderma (Trichoderma spp.), Trichosporon (Trichosporon spp.), Gum (Trichosporium spp.), Rhizochytrium (Ulocladium spp.), Uglaucosporium (Ugla spp.), and Verticillium (Thermocladium spp.).

The microbial infection that can be treated by the peptides of the invention can be selected from any of the bacterial, fungal, parasitic, enveloped viral pathogens of any of which are shown in table 1.

Accordingly, the invention provides the use of a peptide of the invention in the manufacture of a medicament for the treatment of a microbial infection, wherein the microbial infection is a systemic, topical, subcutaneous, cutaneous or mucosal fungal infection.

Mould infections can be classified as systemic or topical infections, meaning infections that are more severe and affect visceral or blood-borne transmission, and topical (dermatophyte) infections meaning infections that are superficial and occur on the skin. In addition, yeast infections can affect the mucosa of the body. Yeast infections can also be systemic (e.g., candidemia and other often fatal disease states). Creams or ointments (topical antifungals) can be used to treat fungal infections on the skin in general. However, systemic infections, yeast infections, or local infections that are not eliminated after treatment with creams or ointments may require treatment with systemic antifungal drugs (oral or intravenous). These drugs are used to treat common fungal infections such as tinea (ringworm) which occurs on the skin or candidiasis (yeast infection, also known as fungal stomatitis) which occurs in the throat, vagina or other parts of the body. Systemic antifungal drugs are also used to treat other deep-seated fungal infections such as histoplasmosis, blastomycosis, and aspergillosis that can affect the lungs and other organs. These drugs are sometimes used to prevent or treat infections in persons whose immune system is weakened, such as bone marrow or organ transplants and HIV-AIDS patients.

Topical or cutaneous fungal infections that do not normally cause death or serious illness are widespread and economically important because they are expensive to treat. Topical or superficial fungal infections may include infections of the skin, hoof leaves, cuticle, nails, and hair. Skin infections are infections of the skin, nails and toenails.

In a preferred aspect of the invention, the fungal infection is onychomycosis. Onychomycosis may be caused by fungi from, but not limited to, the genus Trichophyton (Trichophyton spp.) such as Trichophyton interdigitale or Trichophyton rubrum (Trichophyton rubrum).

The term "onychomycosis" includes, but is not limited to, distal subcategory, superficial white, sub-white, secondary dystrophies, primary dystrophies, endonyx, candida-induced (e.g., onycholysis and chronic mucocutaneous disease) types of onychomycosis. Onychomycosis has been shown to be an important risk factor for a number of serious chronic complications such as acute bacterial cellulitis of the arms/legs and other secondary bacterial infections, and the present invention therefore includes the treatment of these infections.

The peptides of the invention are effective antimicrobial peptides for a variety of pathogenic organisms. However, the peptides of the invention may also be used in the treatment of other disease states including, but not limited to, cystic fibrosis and other disease states associated with mucosal infections such as gastrointestinal infections, urogenital infections or respiratory infections.

The peptides of the invention may also be used in particular for the treatment or prevention of wounds, ulcers and injuries, such as epidermal wounds, e.g. cuts or burns, and disease states associated therewith.

The term "treatment" relates to the effect of a peptide as described herein on the benefit of administration to a patient suffering from an (infectious) disease, including amelioration of the disease state or delay of progression of the disease in the patient.

As used herein, "wound treatment" may include wound healing and related conditions and therapies that promote, enhance or accelerate tissue healing and include post-operative scarring, burns, psoriasis, acceleration of tissue remodeling such as after plastic surgery and organ transplantation.

Thus, another aspect of the invention provides a substrate using or attached to a peptide of the invention. Preferably, the matrix is suitable for use in or delivery to a wound site. Preferably, the matrix allows the peptides of the invention to be transferred from the matrix to the wound bed to achieve their antibacterial effect. The substrate may be a dressing, such as a wound dressing. The dressing may comprise a fabric material or it may be a collagen-like material.

The invention can also be used as or in disinfectants. In this regard, the peptides or pharmaceutical compositions of the present invention may be applied to the surface in need of treatment alone or in combination with other disinfectants. As used herein, a "surface in need of treatment" may be a substrate or medical device as defined herein.

In another aspect, the invention provides a method of treating or preventing a microbial infection in a subject, comprising administering to the subject a therapeutically effective amount of a peptide of the invention.

In a preferred method of the invention, the microbial infection is a fungal infection. In the methods of the invention, the peptide may be applied to the skin or nail of the subject.

The peptides, compositions, or methods described herein can be used to treat mammals, birds, and other animals. Such mammals and birds include humans, dogs, cats, and livestock such as horses, cattle, sheep, goats, chickens, turkeys, and the like. In addition, plants can also be treated using the peptides, compositions or methods of the invention.

When the individual is an animal, the methods of the invention may be used on nail-like parts including hooves, claws and feet, non-exclusively.

In addition to peptide therapy, the methods of the invention may include therapies that enhance penetration of peptides into the nail. The above-described treatments may be facilitated by chemical or physical means. Physical treatments such as etching or etching back of the nail layer may enhance the permeability of the peptides of the invention. Chemically enhancing the nail permeability of the peptides of the invention may be achieved by breaking the physical or chemical bonds of the nail keratin. The penetration of the peptides of the invention may be enhanced by the nail softening agent, which non-exclusively includes urea and salicylic acid, enhancing hydration of the nails to reduce nail density. Compounds containing a sulfhydryl group will disrupt the disulfide bond in the keratin and may lead to instability and enhanced drug permeability. Compounds which non-exclusively include acetylcysteine and mercaptoethanol derivatives and the like may be used in combination with the peptides of the present invention. Other known nail penetration excipients/adjuvants that may be used in combination with the peptides of the invention include methylsulfonylmethane, urea, polyethylene glycol, N- (-2-mercaptopropionyl) glycine, dimethyl sulfone, and 2-N-nonyl-1, 3-dioxolane.

In another aspect, the invention provides a method of treating a wound in an individual comprising applying to the wound of the individual a therapeutically effective amount of a peptide or matrix of the invention.

The peptides of the invention, including salts thereof, may be administered in order to achieve a reduction in at least one symptom associated with an infection, sign or disease, or a reduction in the amount of antibody associated with a sign or disease.

To achieve the desired effect, the peptides, variants thereof, or combinations thereof may be administered in a single dose or in discrete doses, for example, at least about 0.01mg/kg body weight to about 500 to 750mg/kg body weight, at least about 0.01mg/kg body weight to about 300 to 500mg/kg body weight, at least about 0.1mg/kg body weight to about 100 to 300mg/kg body weight, or at least about 1mg/kg body weight to about 50 to 100mg/kg body weight, or at least about 1mg/kg body weight to about 20mg/kg body weight, although other doses may also provide beneficial results. The amount administered may vary depending on a variety of factors including, but not limited to, the peptide selected and its clinical effect, the disease, the weight of the mammal, the physical condition, health, age, whether prevention or treatment is to be achieved, and whether the peptide is chemically modified. These factors can be readily determined by the clinician reviewing empirical data from clinical trials and examining results from preclinical animal models or other test systems available in the art.

The therapeutic agents of the present invention may be administered in a single dose, multiple doses in a continuous or intermittent manner, depending, for example, on the physiological condition of the recipient, whether the administration is for therapeutic or prophylactic purposes, and other factors known to the skilled artisan. The peptides of the invention may be administered substantially continuously over a predetermined period of time, or may be administered in a series of spaced doses. Local administration and systemic administration can be carried out.

To prepare the composition, the peptide is synthesized or otherwise obtained, and if necessary or desired, the peptide can be purified and then lyophilized and stabilized. The peptide is then adjusted to the appropriate concentration and optionally mixed with other reagents. The absolute weight of a given peptide contained in a unit dose can vary widely. For example, about 0.01 to about 2g or about 0.01 to about 500mg of at least one peptide of the invention or a plurality of peptides specific to a particular cell type may be administered. Alternatively, the unit dose can be about 0.01g to about 50g, about 0.01g to about 35g, about 0.1g to about 25g, about 0.5g to about 12g, about 0.5g to about 8g, about 0.5g to about 4g, or about 0.5g to about 2 g.

The daily dosage of the peptides of the invention may also vary. For example, the daily dose can be from about 0.001 g/day to about 100 or 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.1 g/day to about 5 g/day, from about 0.1 g/day to about 2.5 g/day, from about 0.1 g/day to about 2 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, from about 0.5 g/day to about 2 g/day, and from about 0.5 g/day to about 1 g/day.

Thus, one or more suitable unit dosage forms comprising the therapeutic peptides of the invention may be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular, and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary, and intranasal (respiratory) routes. The therapeutic peptides may also be formulated in a lipid dosage form or sustained release (e.g., using microencapsulation, see WO94/07529 and U.S. Pat. No. 4,962,091). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods may include the step of mixing the therapeutic agent with a lipid carrier, a solid matrix, a semi-solid matrix, a finely divided solid carrier, or a combination thereof, and then, if desired, introducing or shaping the product into a desired delivery system.

When the therapeutic peptides of the present invention are prepared for oral administration, they are typically mixed with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. For oral administration, the peptides may be present in the form of a powder, granular formulation, solution, suspension, emulsion or in a natural or synthetic polymer or resin to facilitate absorption of the active ingredient from the chewing gum. The active peptides may also be present in the form of a bolus, electuary or paste. The therapeutic peptides of the invention may also be formulated for sustained release for oral administration, for example, the peptides may be coated, microencapsulated, or placed in a device for sustained delivery. The total active ingredient in such formulations is 0.1 to 99.9% by weight of the formulation.

Pharmaceutical formulations containing the therapeutic peptides of the invention can be prepared by methods known in the art using well-known and readily available starting materials. For example, the peptides may be formulated with conventional excipients, diluents or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols, and the like. Examples of excipients, diluents and carriers suitable for these formulations include buffers, fillers and bulking agents such as starch, cellulose, sugars, mannitol and silicon derivatives. The formulations may also include binding agents such as carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone. The formulation may include a moisturizing agent such as glycerin, a disintegrating agent such as calcium carbonate and sodium bicarbonate. Dissolution retarding agents such as paraffin wax may also be included. Absorption accelerators such as quaternary ammonium compounds may also be included. Surfactants such as cetyl alcohol and glyceryl monostearate may be included. Adsorption carriers such as kaolin and bentonite may be added. Lubricants such as talc, calcium and magnesium stearates and solid polyethylene glycols may also be included. Preservatives may also be added. The compositions of the present invention may also contain thickeners such as cellulose and/or cellulose derivatives and the like. They may also contain gums such as xanthan, guar or carbopol or acacia, or polyethylene glycols, bentonites and montmorillonites, and the like.

For example, a tablet or caplet (caplet) containing the peptide of the present invention may contain a buffering agent such as calcium carbonate, magnesium oxide, and magnesium carbonate. Suitable buffering agents may also include acetates, citrates, borates and phosphates. Caplets and tablets may also contain inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxypropylmethyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatin capsules containing at least one peptide of the invention may contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, titanium dioxide, and liquid carriers such as polyethylene glycols (PEGs) and vegetable oils. In addition, enteric coated capsule tablets or troches containing one or more peptides of the invention are designed to resist disintegration in the stomach and dissolution in the more neutral to alkaline environment of the duodenum.

The therapeutic peptides of the invention may also be formulated as elixirs or solutions for convenient oral administration or as solutions suitable for parenteral administration by routes such as intramuscular, subcutaneous, intraperitoneal or intravenous. The pharmaceutical formulations of the peptides of the invention may also be in the form of aqueous or anhydrous solutions or dispersions, or in the form of emulsions, suspensions or ointments.

Thus, the therapeutic peptides may be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers, or multi-dose containers. As noted above, preservatives may be added to help maintain the shelf life of the dosage form. The active peptides and other ingredients may be formed into suspensions, solutions or emulsions in oily or aqueous media, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active peptides and other ingredients may be in powder form, obtained by sterile isolation of a sterile solid or by lyophilization from solution, which is to be mixed with a suitable carrier, e.g., sterile, pyrogen-free water, before use.

These formulations may contain pharmaceutically acceptable carriers, vehicles and adjuvants well known in the art. For example, in addition to water, the solution may be prepared using one or more physiologically acceptable organic solvents selected from acetone, acetic acid, ethanol, isopropanol, dimethyl sulfoxide, glycol ethers (such as those sold under the name "Dowanol"), polyethylene glycol (polyglycol) and polyethylene glycol (polyethylene glycol), short chain acid C₁-C₄Alkyl esters, ethyl or isopropyl lactate, fatty acid triglycerides (for example under the name "Miglyol "products sold), isopropyl myristate, animal, mineral and vegetable oils and silicones.

Preferably, the pharmaceutical formulation of the therapeutic peptide of the invention may also be in the form of a solvent or diluent containing the peptide. The solvent or diluent may include an acid solution, dimethyl sulfone, N- (-2-mercaptopropionyl) glycine, 2-N-nonyl-1, 3-dioxolane, and ethanol. The solvent/diluent is preferably an acidic solvent, such as acetic acid, citric acid, boric acid, lactic acid, propionic acid, phosphoric acid, benzoic acid, butyric acid, malic acid, malonic acid, oxalic acid, succinic acid or tartaric acid.

More preferably, the solvent is an acetic acid solution. The solvent, such as an acetic acid solution, may be present in the composition at a concentration of less than 1%, 0.5%, 0.25%, 0.1%, 0.05% or 0.01% acid, such as acetic acid.

Another aspect of the invention provides the use of an acid in the manufacture of a medicament for the treatment of microbial infections, in particular fungal infections. The fungal infection may be onychomycosis. Onychomycosis may be caused by a fungus from, but not limited to, the genus Trichophyton (Trichophyton spp.) such as Trichophyton interdigitale or Trichophyton rubrum (Trichophyton rubrum). The acid may be as described above. The preferred acid is acetic acid. Preferably, the acid is provided in solution at a concentration of less than 1%, 0.5%, 0.25%, 0.1%, 0.05% or 0.01% acid, such as acetic acid. The medicaments are generally adapted for topical administration to treat, for example, nails.

The term "active agent" as used hereinafter includes a single peptide of the invention or a combination of peptides as described herein. The term "active agent" may also include a pharmaceutically effective amount of an acid as described herein. The active agents may be administered simultaneously, sequentially or separately. Topical administration is generally preferred.

The active agents may be administered in synergistically effective amounts. Accordingly, the present invention comprises: use of a synergistically effective amount of an active agent, e.g. a peptide of the invention and a pharmaceutically effective amount of an acid as described herein, for the manufacture of a product, e.g. a medicament, for simultaneous, separate or sequential administration of said active agents in the treatment of a microbial infection.

If desired, adjuvants selected from antioxidants, surfactants, other preservatives, film forming agents, keratolytic or exfoliating agents, fragrances, flavoring agents, and coloring agents may be added. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and alpha-tocopherol and derivatives thereof may be added.

The invention also relates to a combination product comprising one or more peptides of the invention and one or more other antimicrobial or antifungal agents, such as amphotericin B, amphotericin B liposome complex (ABCD), liposomal amphotericin B (L-AMB) and liposomal mycotoxins, azoles and triazoles, e.g. voriconazole, fluconazole, ketoconazole, itraconazole, pozaconazole and the like; glucan synthase inhibitors such as caspofungin, micafungin (FK 463) and V-echinocandin (LY 303366); griseofulvin; allylamines, such as terbinafine; flucytosine or other antifungal agents including those described herein. In addition, the invention also relates to the fact that the peptides may be mixed with topical antifungal agents such as ciclopirox olamine, haloprogin, tolnaftate, undecylenate, topical nystatin, amorolfine, butenafine, naftifine, terbinafine and other topical agents.

In addition, the peptides are also suitable for use in formulations of sustained-release dosage forms and the like. The formulation may be composed in a specific manner such that it releases the active peptide, for example, over a certain period of time in a specific part of the intestinal or respiratory tract. The coating, envelope and protective matrix may be prepared from polymeric matrices such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles or waxes. These coatings, envelopes and protective matrices can be used to coat internal devices such as stents, catheters, peritoneal dialysis tubing, drainage devices, and the like.

For topical administration, the active agent may be formulated as known in the art for direct application to the target area. The main customary forms for topical application are creams, milks, gels, powders, dispersions or microemulsions, more or less thickened lotions, impregnated pads, ointments or sticks, aerosol preparations (e.g. sprays or foams), soaps, detergents, soap lotions or soap bars. Other conventional forms for this purpose include wound dressings, applied bandages or other polymeric coverings, ointments, creams, lotions, pastes, gels, sprays and aerosols. Thus, the therapeutic peptides of the invention can be delivered by a patch or bandage for dermal administration. Alternatively, the peptide may be formulated as part of a viscous polymer such as a polyacrylate or an acrylate/vinyl acetate copolymer. For long term applications, it may be desirable to use a microporous and/or breathable backing layer in order to minimize hydration or skin maceration. The backing layer may be of any suitable thickness that provides the desired protective and support functions. Suitable thicknesses are typically 10 to 200 microns.

The topical administration may be in the form of a nail coating or a nail lacquer layer. For example, the antifungal peptide formulation may be administered topically in a solution containing ethyl acetate (NF), isopropanol (USP), and butyl monoester of poly [ methyl vinyl ether/maleic acid ] in isopropanol.

Pharmaceutical formulations for topical administration may comprise, for example, a physiologically acceptable buffered saline solution containing from about 0.001mg/ml to about 100mg/ml of one or more peptides of the invention specific for the indication or disease to be treated, e.g., at a concentration of from about 0.1mg/ml to about 10 mg/ml.

Ointments and creams may be formulated with an aqueous or oily base incorporating suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Active peptides may also be delivered by iontophoresis as disclosed in U.S. patent nos. 4,140,122, 4,383,529 or 4,051,842. The weight percentage of the therapeutic agent of the present invention present in the topical formulation will depend on a variety of factors, but is generally from 0.01% to 95%, typically from 0.1-85% by weight of the total weight of the formulation.

Drops such as eye drops and nasal drops of one or more of the therapeutic peptides may be administered in an aqueous or non-aqueous base which may also contain one or more dispersing, solubilising or suspending agents. Liquid sprays can be pumped out or conveniently delivered from a pressurized pack. Drops may be delivered by simple sealed eye drops, by plastic bottles suitable for drop-wise delivery of liquid contents, or by specially shaped enclosures.

The therapeutic peptide formulation may also be used for topical administration in the mouth or throat. For example, the active ingredient groups may be presented as lozenges comprising a flavoured base, usually sucrose and acacia or tragacanth, pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia, and mouthwashes comprising the composition of the invention in a suitable liquid carrier.

The pharmaceutical formulations of the present invention may include as optional ingredients pharmaceutically acceptable carriers, diluents, solubilizers or emulsifiers, and salts of the type available in the art. Examples of such substances include common saline solutions, such as physiologically buffered saline solution and water. Specific, but non-limiting examples of carriers and/or diluents that may be used in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline at ph 7.0-8.0.

The peptides of the invention may also be administered to the respiratory tract. Accordingly, the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention. Typically, these dosage forms include an amount of at least one agent of the invention effective to treat or prevent the clinical symptoms of a particular infection, indication, or disease. Any statistically significant attenuation of an infection, sign or disease treated according to the methods of the present invention is considered to be a treatment for such infection, sign or disease within the scope of the present invention.

Alternatively, for administration by inhalation insufflation, the compositions may be prepared in the form of a dry powder, for example a powder mix of the therapeutic agent and a suitable powder base such as lactose and starch. The powder compositions may be presented in unit dosage form in capsules or cartridges, or in, for example, gelatin or blister packs, from which they are administered using an inhaler or insufflator, or a metered dose inhaler (see, for example, the pressurized Metered Dose Inhalers (MDIs) and dry powder inhalers disclosed in newina, s.p. in aerogels and the Lung, Clarke, s.w. and Davia, d.eds., pp.197-224, butterworks, London, England, 1984).

When administered as an aerosol or inhalation, the therapeutic peptides of the invention may be administered as an aqueous solution. Thus, other pharmaceutical formulations may contain, for example, a physiologically acceptable buffered saline solution containing from about 0.001mg/ml to about 100mg/ml of one or more peptides of the invention specific for the indication or disease to be treated. Finely divided solid peptides or dry aerosols in the form of nucleic acid particles that are insoluble or not suspended in liquid may also be used in the practice of the invention. The peptides of the invention may also be formulated as dusting powders and contain finely divided particles having an average particle size of about 1 to 5 μm or 2 to 3 μm. Finely divided particles can be prepared by size reduction or screen filtration using techniques well known in the art. The particles may be administered by inhalation of a predetermined amount of a finely divided substance, which may be in powder form. It will be appreciated that the unit content of active ingredient or the ingredients contained in the individual aerosol doses of each dosage form need not in themselves constitute an effective amount for the treatment of a particular infection, indication or disease, since the necessary effective amount can be achieved by administration of multiple unit doses. In addition, an effective amount may be achieved in one administration or in multiple administrations using lower doses than in the dosage form.

For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic peptides of the invention may conveniently be delivered from a nebulizer or a pressurized pack or other suitable device for delivery of an aerosol spray. The pressure packs may contain a suitable compressed gas such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, and the like. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Nebulizers include, but are not limited to, those disclosed in U.S. patent nos. 4,624,251, 3,703,173, 3,561,444 and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from a number of commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ), and American Pharmoseal Co. (Valencia, CA). For intranasal administration, the therapeutic agent may also be administered by nasal drops, liquid sprays, e.g., by a plastic bottle nebulizer or metered dose inhaler. Common nebulizers are Mistometer (Wintrop) and Medihaler (Riker).

In addition, whether used in the disease state or some other disease state, the active ingredient may also be used in combination with other therapeutic agents such as analgesics, anti-inflammatory agents, antihistamines, bronchodilators, and the like.

The present invention also provides screening assays for specific peptides that have low toxicity to ordinary human or other animal cells but have desirable antimicrobial properties such as antifungal (penetrating the cell membrane of the fungus, solubilizing or otherwise killing or inhibiting the growth of the fungus).

Candidate peptides can be obtained from the peptide libraries of the invention described herein. The peptides may also be designed individually or rationally to have specific structural features.

The invention is illustrated by way of example and with reference to the following drawings:

FIG. 1 shows the amino acid sequences of 4 peptides of the invention;

fig. 2 is a histogram showing the growth of the fungus trichophyton intertoe (t.interdigitale) after treatment with the peptide of fig. 1 for (a)4 days and (b)7 days;

fig. 3 is a histogram showing the growth of the fungus trichophyton rubrum (t.rubrum) after treatment with the peptide of fig. 1 for (a)4 days and (b)7 days;

FIG. 4 is a histogram showing the growth of the fungus Candida albicans (Candida albicans) after 24 hours and (b)48 hours of treatment with the peptide of FIG. 1;

FIG. 5 is a histogram showing the results of a dose response experiment of peptide 1 (shown in FIG. 1) to growth of Candida albicans (Candida albicans) 24 hours after treatment;

FIG. 6 is a graph showing the 24-hour survival rate of Candida (Candidaspp.) at different doses of peptide 4 shown in FIG. 1;

FIG. 7 is a graph showing the 24 hour survival rate of 3 different bacterial strains at different doses of peptide 4 shown in FIG. 1;

fig. 8 is a histogram showing the synergistic effect of 0.01% acetic acid on the antifungal (anti trichophyton rubrum (t.rubrum)) activity of peptide 4(1mg/ml) at 3 days of growth;

fig. 9 is a histogram showing inhibition of peptide 4 against trichophyton intertoe (t.interdigital) and trichophyton rubrum (t.rubrum);

fig. 10 is a histogram showing the effect of peptide 3 and peptide 4 on trichophyton intertoe (t.interdigital);

fig. 11 is a histogram showing the effect of acetic acid on the growth of trichophyton intertoe (t.interdigital);

fig. 12 is a histogram showing the effect of polylysine on the growth of t.interdigital;

fig. 13 is a histogram showing the effect of polylysine and polyarginine on the growth of trichophyton rubrum (t.rubrum);

fig. 14 is a histogram showing the inhibition of p.interdigital (t.interdigital) and p.rubrum (t.rubrum) by poly-L-arginine;

fig. 15 is a histogram showing the effect of reduced concentrations of polyarginine on trichophyton rubrum (t.rubrum) and trichophyton intertoe (t.interdigital);

fig. 16 is a histogram showing the effect of trimers on trichophyton rubrum (t.rubrum) growth;

figure 17 is a histogram showing the effect of peptide 4 and NaCl on the growth of trichophyton intertoe (t.interdigital);

FIG. 18 is a graph showing the effect of peptide 4 on Candida albicans (Candidaalbicans) at increasing salt concentrations;

FIG. 19 is a histogram showing the effect of polylysine and polyarginine on Candida albicans (Candidaalbicans) survival;

table 1 lists human bacterial, fungal, parasitic and enveloped viral pathogens that can be treated with the peptides of the invention;

table 2 details the peptides corresponding to the results and the amino acid polymer codes shown in the figures.

The invention is described by reference to the following examples.

Examples

Materials and method reagents

Or produced using solid phase synthesis by contracting with Invitrogen-Evoquest, Carlsbad, CA, USA, or all peptides were obtained from the peptide supplier NeoMPS SA (Strasbourg, France) or Sigma-Aldrich Chemical Company Ltd (Poole, UK). For tests using fungi, the lyophilized peptides were formulated as 1,000 μ g/ml stock solutions in assay buffer. When the experiments in fig. 2 to 8 and fig. 11 were made to specify, acetic acid was added as a solvent to make the final concentration 0.5%.

Pathogens

Strains of T.interdigital (NCPF117) and T.rubrum (NCPF335) were obtained from the National Collection of Pathogenic Fungi, Bristol, and the strains were transferred to a Sabouraud's agar slant and a potato dextrose agar slant at intervals of about one month, and maintained at 30 ℃. Candida albicans (Candida albicans) strain 3179 (obtained from National Collection of TypeCulturs [ NCTC ], Colindale) was fed at 37 ℃ in Oxoid Mueller Hinton Broth. Streptococcus pyogenes (Streptococcus pyogenes) strain 8198, Staphylococcus aureus (Staphylococcus aureus) strain 10642 (methicillin resistant) and E.coli0157 strain 12900 were obtained from NCTC, Colindale and fed in Oxoid Mueller Hinton Broth at 37 ℃.

Fungal growth sensitivity assay

To determine the sensitivity of the fungal strain to each test peptide, the effect of the test peptide on fungal growth was evaluated as follows. Suspensions of conidia and hyphal segments of inter-digital and deep red trichophyton (t.rubrum) were prepared by adding 10ml of fresh Nutrient Glucose Broth (NGB) (Oxoid Nutrient broth containing 2% w/v glucose) to the skew medium and stirring with a spatula. The resulting conidia/hyphal segment suspension was filtered through 2 layers of sterile surgical gauze to remove larger hyphal clumps and agar blocks. To each well of a sterile 96-well microtiter plate to which a total volume of 80. mu.l of nutrient medium (NGB) and an appropriate amount of peptide solution had been added 20. mu.l of this suspension (absorbance at 540nm of about 0.1, corresponding to about 10)⁶Propagules/ml). Control wells are wells with a final assay volume of 100 μ l using only NGB medium and solvent (if applicable, and the same concentration as the peptide sample if used). After 24 hours, 4 days, 7 days incubation at 30 ℃, fungal growth within the plates was monitored by reading the absorbance at 540nm by a Microtek plate reader.

Candida albicans (Candida albicans) survival assay

To determine the sensitivity of the fungal strains to each test peptide, the effect of the test peptide on the survival of Candida albicans (Candida albicans) was evaluated as follows. Cultures of Candida albicans (C.albicans) were grown for 18 to 24 hours and then used beforeStorage at 4 ℃. The overnight-grown fresh culture was centrifuged at 2000x g for 10 minutes and washed with fresh Mueller Hinton Broth, and the number of viable cells was adjusted to 5X 10⁶To 1X 10⁷And/ml. The assay buffer was prepared by adding 100. mu.l of NGB medium to 6.9ml of 10mM sodium phosphate buffer (pH 7.7). To a sterile screw-capped polypropylene vial was added 35. mu.l of assay buffer with or without a concentration of peptide, and 15. mu.l of the Candida albicans (Candidaalbicans) inoculum described above. The vials were incubated in a water bath at 37 ℃ for 2 hours and the number of surviving Candida species (Candida spp.) was determined by serial dilution in sterile Phosphate Buffered Saline (PBS) and plating on 9cm Petri dishes containing oxoid sabouraud's Agar (20 ml). The plates were counted after incubation at 37 ℃ for 18 to 24 hours.

Bacterial survival analysis

Streptococcus pyogenes (Streptococcus pyogenes) strain 8198, Staphylococcus aureus (Staphylococcus aureus) strain 10642 (methicillin resistant) and e.coli0157 strain 12900 (both obtained from NCTC, Colindale) were grown for 18 to 24 hours and then stored at 4 ℃ prior to use. The overnight grown fresh culture was centrifuged at 2000x g for 10 minutes and washed with fresh Mueller Hinton Broth. Each of the 4 peptides was analyzed according to the method of candida albicans (c. Initial number of cells used by the peptide sensitive species for e.coli and s.aureus (s.aureus) was 10⁸Ml and the medium used for counting is Nutrient Agar (Oxoid). Streptococcus pyogenes (srt. pyogenes) did not grow as well on mueller hinton agar as other strains and therefore the starting cell number for these assays was lower than for the other strains, the latter having a starting cell number of 10⁶And/ml. The survival of streptococcus pyogenes (srt. pyogens) was determined using Oxoid trypte Soya Agar instead of nutrentagar.

Results

Peptide 1 to peptide4 inhibition of growth of Trichophyton sp

As described in the materials and methods section, two clinically relevant dermatophyte pathogens trichophyton rubrum (trichophyton rubrum) and trichophyton interdigitale (trichophyton interdigitale) were cultured in growth medium alone (control culture) or growth medium containing 50 μ l/ml of peptide 1, peptide 2, peptide 3 or peptide 4 (see fig. 1). After 4 and 7 days of culture, the growth of trichophyton intertoe (t.interdigital) and trichophyton rubrum (t.rubrum) was evaluated by measuring the optical density (absorbance at 540 nm). Each of the peptides tested significantly inhibited the growth of t.interdigital (fig. 2) and t.rubrum (fig. 3) on days 4 and 7, compared to the control, untreated samples. Control cultures of each test strain continued to grow as indicated by increased OD readings between 4 and 7 days.

Inhibition of Candida growth and survival by peptides 1 through 4

Yeast Candida albicans (Candida albicans) was cultured in growth medium alone (control culture) or containing 50. mu.l/ml or 100. mu.l/ml and 300. mu.l/ml or 500. mu.l/ml of peptide 1, peptide 2, peptide 3 or peptide 4 as described in the materials and methods section. After 24 hours (fig. 4a) and 48 hours (fig. 4b) of culture, the growth of candida albicans (c. albicans) was evaluated by measuring the optical density (absorbance at 540 nm). Each of the test peptides significantly inhibited the growth of candida albicans (c. albicans) in a time and dose dependent manner as compared to the control, untreated, samples. The dose-dependence of growth inhibition was further demonstrated in experiments in which the growth of candida albicans (c.albicans) was assessed optically under control (growth medium alone) conditions or after 24 hours incubation in the presence of peptide 1 from 50 μ g/ml to 500 μ g/ml (fig. 5). In another experiment, the survival of Candida albicans (C.albicans) was evaluated after 18 to 24 hours of culture growth in medium alone (control) or medium containing 1 to 1000. mu.g/ml peptide 4 (FIG. 6). The survival of candida albicans (c. albicans) was decreased in a dose dependent manner by survival counts after 24 hours of culture (fig. 6).

Inhibition of bacterial survival by peptide 4

As described in the materials and methods section, three clinically relevant bacterial pathogens e.coli0157, methicillin-resistant staphylococcus aureus (MRSA) and Streptococcus pyogenes (Streptococcus pyogenes) were exposed to a solution containing a concentration of peptide 4. After 3 hours each bacterial culture sample was transferred to a suitable solid growth medium plate and the number of viable colonies in the control (growth medium only) and treated (growth medium containing peptide 4) samples was evaluated after 18 to 24 hours. After 3 hours of exposure, peptide 4 significantly inhibited the survival of each bacterial strain in a dose-dependent manner compared to the control, untreated culture (figure 7).

Acetic acid enhances the antifungal activity of peptide 4

Since both the control (without peptide) and test (with peptide 1, peptide 2, peptide 3 or peptide 4) media contained 0.5% acetic acid as the peptide solvent in the experiments shown in fig. 2 and 3 (detailed in the materials and methods section), separate experiments were performed to determine whether acetic acid itself could play a role in peptide activity and/or fungal survival. To this end, according to the materials and methods section, growth medium only, growth medium containing 0.01% acetic acid, growth medium containing 1mg/ml peptide 4, and growth medium containing 1mg/ml peptide 4 and 0.01% acetic acid were used to establish trichophyton rubrum (t.rubrum) growth experiments. Fungal growth was determined by OD after 3 days of culture as described above. As expected, peptide 4 inhibited the growth of trichophyton rubrum (t. 0.01% acetic acid alone had no significant effect on the growth of trichophyton rubrum (t.rubrum) (fig. 8), but when included in the medium containing peptide 4, the presence of 0.01% acetic acid significantly inhibited the growth of trichophyton rubrum (t.rubrum) more than 1mg/ml peptide 4 alone.

Peptide 4 growth on interdigital and T.rubrum Suppression of

The inhibitory effect of peptide 4 on the growth of Trichophyton sp was determined by fungal growth analysis based on materials and methods. T.rubrum and t.interdigital were cultured in separate media or media containing 3 different concentrations of peptide 4. Acetic acid was not present in any of the samples. Control medium was used to illustrate the background absorbance of the medium. Fungal growth was determined by OD after 96 hours incubation at 30 ℃ as described previously. As shown in fig. 9, these analyses demonstrated that peptide 4 had inhibitory effects on the growth of both fungi, and that t.interdigital was consistently more susceptible to inhibition by peptide 4 than t.rubrum. The growth of T.interdigital was inhibited at a peptide concentration of 0.55 mg/ml.

Effect of peptide 3 and peptide 4 on the growth of T.interdigital

Peptides 3 and 4 were evaluated for their antifungal potential against trichophyton intertoe (t.interdigitale). Growth inhibition analysis was performed in the absence of acetic acid, depending on the material and method. Since peptides 1 to 3 are very hydrophobic and therefore insoluble, their inhibition of trichophyton spp has previously only been tested in acetic acid as solvent. When trichophyton interdigital (t. interdigital) was grown for 7 days in the presence of peptide 3, peptide 4 or a medium alone without an acetic acid solvent and the growth thereof was measured by OD, it was observed that peptide 4 significantly inhibited the growth of fungi (fig. 10), while peptide 3 showed no inhibitory activity (fig. 10). In the absence of 0.5% acetic acid, the higher activity of cationic peptide 4 over hydrophobic peptide 3 indicates that acetic acid contributes significantly to the previously observed activity of the above hydrophobic peptides.

Effect of acetic acid on the growth of Trichophyton interdigital (T. interdigital)

Inhibition of growth of t.interdigital by acetic acid was evaluated by establishing a fungal growth experiment according to the materials and methods section. Untreated trichophyton intertoe (t.interdigitale) cultures or cultures treated with 3 different concentrations of acetic acid were grown for 96 hours at 30 ℃ (fig. 11). This indicates that there is a significant effect of 0.5% acetic acid at the same concentrations as described above as the peptide 1 to peptide 4 solvents. This experiment, together with the lack of activity of peptide 3 in the absence of acetic acid, showed that peptide 4 is the most active compound against trichophyton spp.

Effect of Poly-L-lysine on the growth of Trichophyton interdigital (T. interdigital)

Because peptide 4 is a highly cationic peptide comprising lysine and arginine residues, the poly-L forms of these amino acids were tested for antifungal activity against t.interdigital using the growth inhibition assay described in detail in materials and methods, in the absence of acetic acid. A control T.interdigital culture, an untreated T.interdigital culture and a Trichophyton sp culture containing poly-L-lysine molecules of 27-100 residues to 100-200 residues in length of 1mg/ml to 50. mu.g/ml were established. The growth of trichophyton interdigitalis (t. intercalary) was evaluated in each culture after 96 hours of culture at 30 ℃. Both sizes of poly-L-lysine inhibited growth of trichophyton (trichophyon spp.) (fig. 12), but molecules 27-100 amino acids in length only had inhibitory activity at higher concentrations (fig. 12), although the larger molecules inhibited growth at all concentrations tested. This indicates that the growth inhibitory effect of lysine on Trichophyton spp is size and dose dependent.

Shadows of poly-L-arginine and poly-L-lysine on growth of T.rubrum Sound box

Then testingMultiple purposepoly-L-arginine andmultiple purposeAntifungal activity of poly-L-lysine against trichophyton rubrum (t. Growth inhibition was determined in the absence of acetic acid, depending on the material and method. In a separate medium, containingMultiple purposepoly-L-arginine (28-86 amino acids in length) andmultiple purposePoly-T.rubrum (T.rubrum) was cultured in a medium containing L-lysine (100-200 amino acids). Non-inoculated control media was established. Growth was maintained and monitored at 30 ℃ for 96 hours.Multiple purposepoly-L-arginine andmultiple purposepoly-L-lysine inhibited the growth of trichophyton rubrum (t. rubrum) (fig. 13). When tested at the same dosage the test solutions were,multiple purposeThe poly-L-arginine has higher inhibitory activity on T.rubrum than that of poly-L-arginineMultiple purposeThe poly-L-lysine is a poly-L-lysine,multiple purposePoly-L-arginine completely inhibited growth at 1mg/ml (FIG. 13).

poly-L-arginine is responsible for trichophyton intermodactyl (T. interdigital) and trichophyton rubrum (T). rubrum) inhibition

Testing by establishing fungal growth experiments according to materials and methods sectionMultiple purposeInhibition of growth of Trichophyton (Trichophtyon spp.) by poly-L-arginine. In a single culture medium or with 3 different concentrationsMultiple purposeTrichophyton rubrum (Trichophyton rubrum) and Trichophyton interdigitatum (Trichophyton interdigitale) were cultured in a medium of poly-L-arginine. Acetic acid was not present in any of the samples. Control medium was used to illustrate the background absorbance of the medium. Fungal growth was determined by OD after 96 hours incubation at 30 ℃ as described previously (figure 14). Polyarginine was observed to be active against both fungi at concentrations as low as 0.55mg/ml (figure 14).

Reduced concentrations (100. mu.g/ml) of polyarginine to Trichophyton rubrum (T. rubrum) and interdigital Effects of Trichophyton (T. Interdigitore)

According to the materials and methods section, polyarginine was tested for inhibition of growth of Trichophyton sp by establishing fungal growth experiments. Untreated cultures of Trichophyton rubrum (Trichophyton rubrum) and Trichophyton interdigital (Trichophyton interdigital) were grown in the absence of acetic acid in any of the samples, or treated with a single concentration of polyarginine (100. mu.g/ml). Control medium was used to illustrate the background absorbance of the medium. Fungal growth was determined by OD after 96 hours incubation at 30 ℃ as described previously (figure 15). The decrease in concentration resulted in a loss of activity, indicating the dose effect of polyarginine on Trichophyton spp.

Effect of peptide trimer (3 amino acids) on the growth of Trichophyton rubrum (T. rubrum)

Peptide trimers of poly-L-lysine, poly-L-arginine, poly-L-histidine and poly-L-tryptophan were tested for their activity on the growth of trichophyton rubrum (t. Growth inhibition was established according to materials and methods and was either not treated with trichophyton rubrum (t. rubrum) or exposed to 2mg/ml of each trimer. Cultures were fed at 30 ℃ for 96 hours. Fungal growth was measured by OD and the results were expressed as a percentage of growth in untreated cultures (figure 16). poly-L-arginine is the most active peptide against t.rubrum, and only a polypeptide containing 3 amino acids is required to cause a significant reduction in the growth of t.rubrum.

Effect of peptide 4(1.2mg/ml) and NaCl on the growth of T.interdigital

The effect of different salt concentrations on the anti-trichophyton interdigital (t.interdigital) antifungal activity of peptide 4 was investigated. A growth inhibition assay for trichophyton interdigital (t.interdigital) was established according to materials and methods in the absence of acetic acid. The cultures were either not treated or exposed to peptide 4 and NaCl at a concentration of 100mM to 500 mM. Trichophyton intertoe (t. interdigital) was fed at 30 ℃ for 96 hours and growth was assessed by OD as previously described (figure 17). Salt concentrations near or above physiological conditions did not affect the antifungal activity of peptide 4 (fig. 17). Antimicrobial activity has been well reported with respect to low salt concentrations to inhibit endogenous beta-defensins.

Effect of peptide 4 on Candida albicans (Candida albicans) at high salt concentrationSound box

After 2 hours incubation at 37 ℃ with different concentrations of peptide 4, the survival of candida albicans (C. albicans) was assessed as described in methods and materials. Two concentrations of NaCl were introduced into the growth medium to determine the effect of physiological conditions and very high salt conditions (known to inhibit the activity of endogenous β -defensin peptides). Significant killing activity of peptide 4 was observed under very high salt conditions (fig. 18). When increasing the concentration of peptide 4, it was observed that the effect of higher salt concentration was reduced (fig. 18). Thus, the salt did not inhibit the antifungal activity of peptide 4.

Poly-L-lysine, poly-D-lysine and poly-D-arginine against Candida albicans (Candida albicans) Activity

The antifungal activity of poly-L-arginine on lysine and poly-L-lysine on poly-D-lysine was evaluated to determine whether any of these peptide variants showed enhanced activity against Candida albicans (Candida albicans). Candida spp was incubated for 2 hours at 37 ℃ in the presence of 100. mu.g/ml, 1mg/ml and 10mg/ml of poly-D-lysine, poly-L-lysine and poly-L-arginine as described in the materials and methods section. Survival was assessed as described previously and showed that the antifungal activity of poly-L-arginine was higher than that of poly-L-lysine (fig. 19). It has also been shown that poly-D-lysine has very similar antifungal activity to poly-L-lysine.

TABLE 1

Non-exclusive table of human bacterial pathogens

TABLE 1 (continuation)

Non-exclusive table of human fungal pathogens

TABLE 1 (continuation)

Non-exclusive table of human parasitic pathogens

TABLE 1 (continuation)

Non-exclusive list of human enveloped viral pathogens

Coating the virus:viruses with outer lipoprotein biplasma obtained by budding from host cell membranes

TABLE 2

Amino acid polymer codes

KKK-L-lysine trimer

RRR-L-arginine

HHH-L-histidine

WWW-L-Tryptophan

pK 3-14-poly-L-lysine-HBr 500-2,000Da (3-14aa)

pK 7-27-poly-L-lysine-HBr1,000-4,000 Da (7-27aa)

pK 100-200-poly-L-lysine-HCl15,000-30,000 Da (100-200aa)

pKd 27-100-poly-D-lysine-HBr4,000-15,000 Da (27-100aa)

pR 28-86-poly-L-arginine-HCl 5,000-15,000(28-86aa)

Claims (24)

1. A peptide, peptide variant thereof or pharmaceutically acceptable salt thereof for use as a medicament in the treatment of a microbial infection, wherein the peptide comprises from 3 to 200 lysines.

2. A peptide or peptide variant as claimed in claim 1 wherein the peptide comprises 3 to 100 lysines.

3. A peptide or peptide variant as claimed in claim 1 wherein the peptide comprises 27 to 200 lysines.

4. A peptide or peptide variant as claimed in claim 1 wherein the peptide comprises 100 to 200 lysines.

5. The peptide or peptide variant of any of claims 1-4, wherein the peptide is acyclic.

6. The peptide or peptide variant of any of claims 1-5, wherein the microbial infection is caused by a bacterial, parasitic, protozoal, viral, or fungal pathogen.

7. The peptide or peptide variant of claim 6, wherein the bacterial pathogen is from a bacterial genus selected from the group consisting of: staphylococci (Staphylococcus spp.), such as Staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis (Staphylococcus epidermidis); enterococcus (Enterococcus spp.) such as Enterococcus faecalis (Enterococcus faecium); streptococcus pyogenes (Streptococcus pyogenes); listeria (Listeriaspp.); pseudomonas spp (Pseudomonas spp.); mycobacteria (Mycobacterium spp.) such as Mycobacterium tuberculosis (Mycobacterium tuberculosis); enterobacter (Enterobacter spp.); campylobacter sp.); salmonella (Salmonella spp.); streptococcus (Streptococcus spp.) such as Streptococcus Group a or Group B (Streptococcus Group a or B), Streptococcus pneumoniae (Streptococcus pneumoniae); helicobacter sp, such as Helicobacter pylori (Helicobacter pylori); neisseria species (Neisseria spp.), such as Neisseria gonorrhoeae (Neisseria gonorrhea), Neisseria meningitidis (Neisseria meningitidis); borrelia burgdorferi (Borrelia burgdorferi); shigella (Shigella spp.), for example Shigella flexneri; escherichia coli (Escherichia coli); haemophilus (Haemophilus spp.) such as Haemophilus influenzae (Haemophilus influenzae); chlamydia species (Chlamydia spp.) such as Chlamydia trachomatis (Chlamydia trachomatis), Chlamydia pneumoniae (Chlamydia pneumoniae), Chlamydia psittaci (Chlamydia psittaci); francisella tularensis (Francisella tularensis); bacillus (Bacillus spp.) such as Bacillus anthracis (Bacillus anthracensis); clostridia (Clostridium spp.) such as Clostridium botulinum (Clostridium botulinum); yersinia spp, such as Yersinia pestis (Yersinia pestis); treponema spp; burkholderia spp, for example Burkholderia rhinoceros (Burkholderia mallei) and Burkholderia pseudorhinoceros (B pseudomallei).

8. The peptide or peptide variant of claim 6, wherein the viral pathogen is from a virus selected from the group consisting of: human immunodeficiency virus (HIV1& 2); human T cell leukemia virus (HTLV1& 2); ebola virus; human papilloma viruses (e.g., HPV-2, HPV-5, HPV-8, HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54 and HPV-56); papovavirus; a rhinovirus; poliovirus; herpes virus; an adenovirus; epstein Barr virus; an influenza virus; hepatitis b virus and hepatitis c virus; a smallpox virus; rotavirus or SARS coronavirus.

9. The peptide or peptide variant of claim 6, wherein said parasitic pathogen is from a parasitic pathogen selected from the group consisting of: trypanosomes (Trypanosoma spp.) (Trypanosoma cruzi), Trypanosoma brucei (Trypanosoma brucei), Leishmania (Leishmania spp.), Giardia (Giardia spp.), trichomonas (trichomonas spp.), Entamoeba (Entamoeba spp.), genistein-grisea (Naegleria spp.), echinospora (Acanthamoeba spp.), hematophagous (Schistosoma spp.), Plasmodium (Plasmodium spp.), cryptosporidium (cryptosporidium spp.), microsporidium sp.), microsporidium (trichomonas spp.), microsporum (Isospora spp.), Trypanosoma microsporum (Plasmodium spp.), microsporidia (trichomonas), trichomonas (trichomonas spp.), and trichomonas (trichomonas).

10. The peptide or peptide variant of claim 6, wherein the fungal pathogen is from a fungal pathogen selected from the genera consisting of: candida (Candida spp.) (e.g. Candida albicans), Epidermophyton (epimorphon spp.), ectochytrium (Exophiala spp.), Microsporum (Microsporum spp.)), trichophyton (trichophyton spp.) (e.g. trichophyton rubrum (t. rubrum) and trichophyton interdigitatum), trichophyton (tineum (tinum sp.), Aspergillus (Aspergillus spp.), blastomyceliophyton (trichophyton spp.)), coccidiodes (coccoida spp.) (Candida spp.), Cryptococcus (Cryptococcus spp.), Histoplasma (trichophyton spp.) (plasmon. sporus, trichophyton (trichophyton spp.) (chlamydospora), trichophyton (trichophyton. sp.), trichophyton (trichophyton spp.) (Candida spp.)), trichophyton (trichophyton. sporus (trichophyton sp.) (chlamydospora), trichophyton (trichophyton sp.) (Candida spp.) (plasmogen (trichophyton), trichophyton (trichophyton spp.) (Candida spp.) (plasmogen (trichophyton (septophomalop.)), trichophyton (trichophyton spp.)(s), trichophyton spp.) (plasmogen (trichophyton) and trichophyton (trichophyton spp.)) sp.)(s), trichophyton (trichophyton sp.)(s), trichophyton (purpurum (trichophyton (p.)) a), trichophyton (trichophyto, Actinomadura spp, lepidoptera spp, Chrysosporium spp, colletotrichum spp, Beauveria spp, Chrysosporium spp, otosporium spp, Conidiobolus spp, kummer silverwood spp, Fusarium spp, Geotrichum spp, zygosporium spp, leguminosarum spp, phyllosphere spp, phytophthora spp, nospora spp, Rhizomucor spp, rhizopus (Rhizomucor spp), rhizopus (rhizopus spp), Rhodotorula spp, Saccharomyces (Saccharomyces spp), actinomycete (scoposporium spp), scoparia (scopulopsis spp), sporotrichum (Sporobolomyces spp), cephalospora (synphastrum spp), Trichoderma (Trichoderma spp), sporotrichosporium (trichosporium spp), gingival cladosporium (ulocladosporium spp), nigrospora (usage spp), verticillium (verticillium spp).

11. The peptide or peptide variant of claim 10, wherein the pathogen is Trichophyton spp.

12. The peptide or peptide variant of claim 11, wherein the pathogen is Trichophyton interdigital.

13. The peptide or peptide variant of claim 11, wherein the pathogen is Trichophyton rubrum (Trichophyton rubrum).

14. The peptide or peptide variant of claim 10, wherein the fungal infection is onychomycosis.

15. A peptide or peptide variant as claimed in any of claims 1 to 5 wherein the microbial infection is a mucosal infection.

16. A peptide or peptide variant as claimed in claim 15 wherein the mucosal infection is cystic fibrosis.

17. A composition comprising a pharmaceutically effective amount of at least one peptide or peptide variant of any one of claims 1 to 16, a pharmaceutically acceptable carrier, excipient or diluent, for use as a medicament in the treatment of a microbial infection.

18. The composition of claim 17, wherein the composition comprises at least two peptides or peptide variants of any one of claims 1-16, wherein the peptides are different.

19. The composition of claim 17 or 18, wherein the excipient or diluent is selected from the group consisting of an acid, dimethyl sulfone, N- (2-mercaptopropionyl) glycine, 2-N-nonyl-1, 3-dioxolane, and ethanol.

20. The composition of claim 19, wherein the excipient or diluent is an acid selected from the group consisting of acetic acid, citric acid, boric acid, lactic acid, propionic acid, phosphoric acid, benzoic acid, butyric acid, malonic acid, malic acid, oxalic acid, succinic acid, or tartaric acid.

21. The composition of claim 20, wherein the acid is acetic acid.

22. The composition of claim 21, wherein the concentration of acetic acid in the composition is less than 1%, 0.5%, 0.25%, 0.1%, 0.05%, or 0.01%.

23. A substrate, a peptide or peptide variant as claimed in any one of claims 1 to 16 or a substrate as claimed in claim

17-22 is adhered to or coated on the substrate.

24. A matrix according to claim 23, wherein the matrix comprises a fabric material or a collagen material.