HK1119390B - Novel antimicrobial peptides with heparin binding activity - Google Patents

Description

Novel antimicrobial peptides with heparin binding activity

The present application is a divisional application of the application having the title "novel antimicrobial peptide having heparin-binding activity" of application No. 200480013728.2, filed 5/19/2004.

Technical Field

The present invention relates to an antimicrobial peptide with heparin binding activity derived from an endogenous mammalian protein substantially free of antimicrobial activity and having from 10 to 36 amino acid residues, wherein said protein is selected from the group consisting of laminin isoforms (laminin isoforms), complement factor C3(complement factor C3), histidine-rich glycoproteins and kininogen, wherein the antimicrobial peptide comprises at least 4 amino acid residues selected from the group consisting of K, R and H. The invention also relates to pharmaceutical compositions comprising said antimicrobial peptides and to the use of antimicrobial peptides and/or antimicrobial/pharmaceutical compositions.

Background

The immune system of mammals such as humans successfully overcomes several infections. However, in some cases, bacteria, fungi or viruses are not always cleared, and this may cause local or systemic acute infections. This is a problem that requires significant attention in perinatal, burn or intensive care units, and in immunocompromised individuals. In other cases, a continuous bacterial persistence of the epithelial surface may cause or exacerbate chronic disease. In humans, chronic skin ulcers, atopic dermatitis and other types of eczema, acne or urogenital infections may be exemplified.

Symptomatic infections can be treated with a variety of drugs. It may also combat some diseases by, for example, vaccines. However, vaccines are not always the best treatment option and no vaccine is available for some microorganisms. When protection is not available, treatment of the disease is sought. Typically, treatment is performed by using antibiotics that kill the microorganisms. However, during the past years, several microorganisms have begun to resist antibiotics. Most likely, the resistance problem will increase in the near future. In addition, several individuals have already developed allergic reactions to antibiotics, thus reducing the possibility of effective use of some antibiotics.

Epithelial surfaces of various organisms are continuously exposed to bacteria. During the last years, it has been recognized that the natural immune system based on antibacterial peptides plays an important role in the initial bacterial clearance at susceptible biological borders (Lehrer, R.I., and Ganz, T. (1999) Curr Opin Immunol 11: 23-27, Boman, H.G. (2000) Immunol Rev.173, 5-16). Antimicrobial peptides kill bacteria by penetrating the bacterial membrane, so the absence of specific molecular microbial targets minimizes resistance development.

Several antimicrobial peptides and proteins unrelated to the peptides described herein are known in the art.

US6,503,881 discloses cationic peptides which are indolicidin analogues for use as antimicrobial peptides. The cationic peptides are derived from different species, including animals and plants.

US5,912,230 discloses antifungal and antibacterial histidine-based peptides. The peptides are based on defined portions of the amino acid sequence of natural human histatin and methods of treatment of fungal and bacterial infections are disclosed.

US5,717,064 discloses methylating lysine-rich lytic peptides. The cleavage peptide is resistant to trypsin digestion and is non-native. The lytic peptides are suitable for in vivo administration.

US5,646,014 discloses antimicrobial peptides. The peptide is isolated from an antimicrobial fraction derived from silkworm hemolymph. The peptides show excellent antimicrobial activity against several microbial strains such as Escherichia coli, Staphylococcus aureus and Bacillus cereus.

McCabe et al, j.biol.chem.277: 27477-27488, 2002 describes a 37kDa antimicrobial chemotactic protein, azurocidin, which contains the heparin binding consensus motifs XBBXBX and XBBBBXXBX.

WO2004016653 discloses peptides based on the sequence azurocidin 20-44. The peptide contains ring structures linked by disulfide bonds.

US6495516 and related patents disclose peptides based on the bactericidal 55kDa protein, bactericidal/permeability increasing protein (BPI). The peptide has antimicrobial effect and has heparin and LPS neutralizing effect.

WO01/81578 discloses a large number of sequences which encode G-coupled protein-receptor related polypeptides which can be used for a large number of diseases.

WO00/27415 discloses peptides suitable for inhibiting angiogenesis. The peptides are analogs of high molecular weight kininogen 5. BLASTp searches show sequences such as kininogen that are conserved or similar across species, with no indication of the function of such conserved regions or whether they function as small peptides at all.

Currently, over 700 different antimicrobial peptide sequences are known (www.bbcm.univ.trieste.it/. about.tossi/search. htm), including cecropin, defensin magainin, and cathelicidins.

Despite the tremendous number of antimicrobial peptides available today, there is an increasing need for new and improved antimicrobial peptides. Antimicrobial peptides can be used to combat or tolerate microorganisms that are resistant to antibiotics and/or other antimicrobial agents. Furthermore, there is a need for new antimicrobial peptides which are non-allergenic when introduced into mammals such as humans. Bacteria have encountered endogenously produced antimicrobial peptides during evolution without significant induction of resistance.

Disclosure of Invention

According to a first aspect, the present invention relates to an antimicrobial peptide with heparin binding activity derived from an endogenous mammalian protein substantially free of antimicrobial activity and having from 10 to 36 amino acid residues, said protein being selected from the group consisting of laminin isoform, complement factor C3, histidine-rich glycoprotein and kininogen, wherein the antimicrobial peptide comprises at least 4 amino acid residues selected from the group consisting of K, R and H.

By providing such an antimicrobial peptide, the risk of allergic reactions to this antimicrobial peptide may be reduced due to the fact that the peptide is derived from endogenous proteins and/or peptides. By using short peptides, the stability of the peptides can be increased and the production costs can be reduced compared to longer peptides and proteins, and thus the present invention is economically advantageous. Andersson et al, Eur J Biochem, 2004, 271, as published after the priority date of this application: 1219-1226, the present invention results from the following findings: peptides having heparin-binding motifs derived from non-antimicrobial endogenous proteins showed antimicrobial activity. In general, the literature has well documented the structural prerequisites for heparin binding and the presence of heparin binding motifs in a variety of proteins. This component includes various laminin isoforms, fibronectin, coagulation factors (coagulation factors), growth factors, chemokines, histidine-rich glycoproteins, kininogens, and many other molecules (see, Andersson et al, (2004) Eur J Biochem 271; 271: 1219-26 and references cited therein), none of which are naturally antimicrobial.

The antimicrobial peptides and corresponding antimicrobial/pharmaceutical compositions of the present invention provide peptides and compositions that facilitate effective prevention, reduction, or elimination of microorganisms. The likelihood of combating or tolerating antibiotic-resistant microorganisms may thus be increased. Furthermore, mammals allergic to commercially available antimicrobial agents can be treated. By providing an antimicrobial/pharmaceutical composition derived from endogenous proteins, the likelihood of an allergic reaction to these particular peptides in a mammal may be reduced or even eliminated. This allows the antimicrobial/pharmaceutical compositions of the present invention to be used in several applications where the antimicrobial/pharmaceutical compositions contact a mammal as a medicament or as an additive to prevent infection.

In addition, the use of short peptides improves bioavailability. Moreover, the use of structurally different heparin-binding antimicrobial peptides with specific or preferential action on gram-negative and gram-positive bacteria or fungi enables specific targeting of various microorganisms, thereby minimizing the occurrence of resistance and ecological problems. The risk of additional ecological stress by new antibiotics is further reduced by supplementing peptides already present in mammals. Finally, these formulations may also enhance the action of endogenous antimicrobial peptides.

According to a second aspect, the present invention relates to an antimicrobial/pharmaceutical composition comprising one or more antimicrobial peptides as described above and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.

According to a third aspect, the present invention relates to the use of the antimicrobial peptides and/or antimicrobial/pharmaceutical compositions described hereinafter.

The antimicrobial peptides of the present invention add to the list of antimicrobial agents that facilitate the selection for various applications to prevent, reduce, or eliminate microorganisms, including but not limited to microorganisms that attack or infect mammals such as humans.

Drawings

FIGS. 1A-C are graphs demonstrating the antibacterial effect of peptides against Enterococcus faecalis (Enterococcus faecalis).

FIGS. 2A and B are culture dishes illustrating radial diffusion assays using a panel of highly active peptides.

FIGS. 3A-C are graphs and tables depicting the antibacterial effect of histidine-rich peptides.

FIGS. 4A-H are electron micrographs showing analysis of Pseudomonas aeruginosa (Pseudomonas aeruginosa) treated with an antimicrobial peptide.

FIGS. 5A-C are photographs showing the heparin binding activity of peptides derived from complement C3, histidine-rich glycoprotein and kininogen.

FIG. 6 is a photograph illustrating the purification of histidine-containing antimicrobial fragments on a nickel-agarose gel.

Detailed Description

Definition of

In the context of the present application and invention, the following definitions apply:

the term "nucleotide sequence" means a sequence having two or more nucleotides. The nucleotides may be of genomic DNA, cDNA, RNA origin, semisynthetic or synthetic origin, or mixtures thereof. The term encompasses both single-stranded and double-stranded forms of DNA or RNA.

The term "antimicrobial peptide" means a peptide comprising from about 10 to about 36 amino acid residues, having antimicrobial and heparin binding activity, and derived from an endogenous mammal that does not naturally have an antimicrobial effect. An "antimicrobial peptide" prevents, inhibits, reduces or destroys microorganisms. Antimicrobial activity can be determined, for example, by the methods of examples 2, 4 or 5.

The term "heparin-binding affinity" means a peptide that binds heparin, either directly or indirectly. For example, heparin binding activity can be determined by the method in example 7. The antimicrobial peptides of the invention, which show affinity for heparin, also bind dermatan sulfate. Thus, the heparin-binding antimicrobial peptide also interacts with the endogenous glycosaminoglycan dermatan sulfate.

The term "amphiphilic" means that hydrophilic and hydrophobic amino acid residues are distributed along opposite sides of an alpha-helical structure, beta-strand, linear, cyclic, or other secondary conformation, which causes one side of the molecule to be predominantly charged and the other side to be predominantly hydrophilic. The degree of peptide amphiphilicity can be assessed by mapping the amino acid residue sequence through various web-based algorithms, such as those found on http:// us. expasy. org/cgi-bin/protscale. pl. The distribution of hydrophobic residues can be observed by helical wheel mapping (helical wheel diagram). A secondary structure prediction algorithm, such as GORIV, may be found at www.expasy.com..

The term "cationic" means a molecule having a net positive charge in the pH range of about 4 to about 12.

The term "microorganism" means any living microorganism. Examples of microorganisms are bacteria, fungi, viruses, parasites and yeasts.

The term "antimicrobial agent" means any agent that prevents, inhibits or destroys microorganisms. Examples of Antimicrobial agents can be found in The Sanford Guide to Antimicrobial Therapy (32 th edition, Antimicrobial Therapy, Inc, US).

In the context of the present invention, amino acid names and atom names are used based on IUPAC nomenclature (IUPAC nomenclature and symbols for amino acids and peptides (residue names, atom names, etc.), Eur J biochem., 138, 9-37(1984) and corrections in Eur J biochem., 152, 1 (1985)) as defined by Protein DataBank (PNB) (www.pdb.org). The term "amino acid" means an amino acid selected from alanine (Ala or a), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) or derivatives thereof.

Detailed description of the invention

Antimicrobial peptides

The present invention relates to an antimicrobial peptide having heparin binding activity derived from an endogenous mammalian protein having substantially no antimicrobial activity and having from 10 to 36 amino acid residues, wherein the antimicrobial peptide comprises at least 4 amino acid residues selected from the group consisting of K, R and H. Two of these amino acid residues may be contiguous. As reported by Margalit et al, 1993J Biol Chem268, 19228-31, about 20 between B amino acid residuesThe distance of (a) constitutes a prerequisite for heparin binding, irrespective of the peptide conformation. The use of short peptides increases bioavailability (e.g., by increasing skin penetration) and reduces production and purification costs as compared to longer peptides or proteins. The antimicrobial peptides of the present invention are a complement to the antimicrobial peptides commercially available today and increase the potential to combat microorganisms that are resistant and/or resistant to existing antimicrobial agents. Novel peptides that are not allergenic to the mammal on which the peptide is based can be identified by deriving novel antimicrobial peptides from endogenous non-antimicrobial proteins.

Moreover, knowledge of the increasing effects of peptides and their dependence on various salt and ionic environments has enabled the design of specific compositions that enhance and control the effects of peptides. Peptides tailored to act on fungi would be more advantageous to target specific diseases such as yeast infection on the mucosa without significantly affecting the bacterial ecology at these sites. The fact that antimicrobial peptides act on bacterial cell membranes suggests that they may act synergistically with antibiotics. Thus, the combination of antibiotics and peptides may have therapeutic advantages. Finally, there is also a need for low cost non-allergenic antimicrobial agents for use in different types of products where microbial growth is to be prevented.

Furthermore, the use of structurally different short heparin-binding antimicrobial peptides with specific or preferential action on gram-negative and gram-positive bacteria or fungi also enables specific targeting of various microorganisms, thereby minimizing resistance problems and ecological problems. By supplementing peptides already present in the organism, the risk of additional ecological stress by new antibiotics is further reduced. The introduction of agents that increase the effect of the peptide may allow the exogenously supplied peptide to be localized and enhanced, which may further minimize the risk of side effects of the peptide outside the treatment area, such as the risk of resistance induction. Finally, these formulations may also enhance the action of endogenous antimicrobial peptides. If antimicrobial peptides are developed to combat microorganisms in humans, the endogenous antimicrobial peptides are derived from endogenous proteins in humans. The same principles apply to other animals such as horses, cattle, pigs or poultry. Antimicrobial peptides may be based on the structure of peptides and/or proteins present in plasma, blood, connective tissue and constituent cells, and may be derived from heparin-binding proteins; laminin isoforms, von Willebrand factor, vitronectin, protein C inhibitors, fibronectin, coagulation factors, growth factors, chemokines, histidine-rich glycoproteins, kininogen, or complement factor C3.

The antimicrobial peptides of the invention have a binding affinity (Kd) for heparin of about 10nM to about 20. mu.M.

The peptide may have a size of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. The length and sequence of the peptide will depend on the source of the antimicrobial peptide and the microorganism to be combated, as well as whether the peptide is to be used to prevent, inhibit, reduce or destroy microorganisms and in what environment the microorganisms are present, and in what environment the antimicrobial peptide will encounter after administration.

According to a first embodiment, the invention relates to antimicrobial peptides based on kininogen proteins or histidine-rich glycoproteins, wherein at least 20% of the amino acid residues are H. The antimicrobial peptide may comprise more than 30, 40 or even 50% H, R and/or K amino acid residues. In particular examples, 1, 2, 3, 4, 5 or 6 amino acid residues are H. For example, the antimicrobial peptide may be selected from SEQ ID NOs: 1. 2, 3 and 4. These peptides are derived from the heparin-binding domain of the non-antimicrobial proteins kininogen and histidine-rich glycoproteins, respectively, and are rich in H residues.

According to another embodiment, the invention relates to complement factor protein-based antimicrobial peptides. For example, the antimicrobial peptide may be selected from SEQ ID NOs: 5. 6 and 7. SEQ ID NO: 5. peptides 6 and 7 are derived from a defined helical segment of the complement factor C3 molecule. The helical region of the C3a molecule of C3 origin is defined by fragments 19-28 (represented by SEQ ID NO: 5) and 47-70 (represented by SEQ ID NOS: 6 and 7), as demonstrated by Hugli and colleagues (Chazin et al, (1988) Biochemistry27, 9139-48, Hugli, Current topics in Microbiology and Immunology, 1989, 153, 181-208). The whole protein C3 did not exert antimicrobial effect. The heparin binding and antimicrobial capacity of peptide fragments derived from C3 have recently been disclosed (Andersson et al, Eur J Biochem, 2004, 271; 271: 1219-.

According to a third embodiment, the invention relates to an antimicrobial peptide derived from laminin. For example, the antimicrobial peptide may be selected from SEQ ID NOs: 8. 9, 10, 11, 12, 13, 14, 15 and 16. Laminin alpha chain LG domains consist of 5 (1-5) LG modules that have been identified as binding sites for heparin and other cell surface receptors (timpl., et al, MatrixBiol, 2000, 19, 309-317). These modular proteins are synthesized during developmental processes such as wound healing, and proteolytic processing of LG modules has been described to occur during these events. The antimicrobial function of heparin-binding epitopes of LG modules has recently been described, not previously disclosed (Andersson et al, Eur J Biochem, 2004, 271; 271: 1219-1226).

Although peptides are derived from endogenous proteins, they can be produced in semisynthetic or synthetic peptide forms and in microorganisms.

The antimicrobial peptide may be extended with one or more amino acid residues, such as 1-100 amino acid residues, 5-50 amino acid residues, or 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 amino acid residues. Such added amino acids may replicate the sequence of the contiguous antimicrobial peptide sequence derived from the non-antimicrobial protein. The amount added depends on which microorganism is to be combated, the stability, toxicity of the peptide, in which product the mammal to be treated or the peptide should be treated and on which peptide structure the antimicrobial peptide is based. The number of amino acid residues to be added to the peptide also depends on the choice of production, e.g. expression vector and expression host, and the choice of preparation of the antimicrobial/pharmaceutical composition. The extension may be in the N-or C-terminal portion of the antimicrobial peptide or in both terminal portions as long as the extension does not disrupt the antimicrobial effect of the peptide. The antimicrobial peptide may also be a fusion protein, wherein the antimicrobial peptide is fused to another peptide.

In addition, the antimicrobial peptide may be operably linked to other known antimicrobial peptides or other substances, such as other peptides, proteins, oligosaccharides, polysaccharides, other organic compounds, or inorganic substances. For example, the antimicrobial peptide may be coupled to a substance that prevents degradation of the antimicrobial peptide in a mammal before the antimicrobial peptide inhibits, prevents, or destroys the life of a microorganism.

Thus, the antimicrobial peptide may be modified by amidation or esterification at the C-terminal portion of the antimicrobial peptide and acylation, acetylation, pegylation, alkylation, etc. at the N-terminal portion.

Alternatively, peptides derived from functional antimicrobial fragments of non-antimicrobial whole proteins may be modified by 1 to 6 amino acid substitutions.

Examples of microorganisms that are inhibited, prevented or destroyed by antimicrobial peptides are bacteria, including gram-positive and gram-negative bacteria such as enterococcus faecalis (enterococcus faecium), escherichia coli (escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Proteus mirabilis (Proteus mirabilis), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus pyogenes (Streptococcus pyogenenes), staphylococcus aureus (staphylococcus aureus), viruses, parasites, fungi, and yeasts such as Candida albicans (Candida albicans) and Candida parapsilosis (Candida parapsilosis).

The antimicrobial peptides may be obtained from natural sources, such as from human cells, c-DNA, genomic clones, or chemically synthesized, or obtained as expression products from cellular sources by recombinant DNA techniques.

Antimicrobial peptides can be synthesized using standard chemical methods, including by automated procedural synthesis. In general, peptide analogs are synthesized according to standard solid-phase Fmoc protection strategies using HATU (N- [ dimethylamino-1H-1, 2, 3-triazolo [4, 5-B ] pyridin-1-ylmethylene ] -N-methylmethanamium hexafluorophosphate N-oxide) as a coupling agent or other coupling agents such as HOAt-1-hydroxy-7-azabenzotriazole. Peptides are cleaved from solid phase resins with trifluoroacetic acid containing suitable scavengers which also deprotect the side chain functionalities. The crude peptide was further purified by preparative reverse phase chromatography. Other purification methods such as partition chromatography, gel filtration, gel electrophoresis or ion exchange chromatography may be used. Other synthetic techniques known in the art, such as tBoc protection strategies, or different coupling reagents, etc., may be utilized to generate equivalent peptides.

Alternatively, synthetic peptides can be produced recombinantly (see, e.g., U.S. Pat. No. 5,593,866). A variety of host systems are suitable for the production of peptide analogues, including bacteria such as e.coli (e.coli), yeasts such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) or pichia pastoris (pichia), insects such as Sf9 and mammalian cells such as CHO or COS-7. There are many expression vectors available for each host, and the present invention is not limited to any one of them as long as the vectors and the host can produce the antimicrobial peptide. Vectors and methods for cloning and expression in E.coli (E.coli) can be found, for example, in Sambrook et al ((Molecular cloning., analytical Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1987) and in Ausubel et al (Current Protocols in Molecular Biology, Greene Publishing Co., 1995).

Finally, the peptides can be purified from plasma, blood, various tissues, and the like. The peptide may be endogenous or produced following enzymatic or chemical digestion of purified protein. For example, heparin binding proteins may be trypsinized and the resulting antibacterial peptides further isolated on a larger scale.

The DNA sequence encoding the antimicrobial peptide is introduced into a suitable expression vector for the host. In a preferred embodiment, the gene is cloned into a vector to produce a fusion protein. To facilitate isolation of the peptide sequence, the peptide and fusion partner are linked using amino acids that are susceptible to chemical cleavage (e.g., CNBr) or enzymatic cleavage (e.g., V8 protease, trypsin). For expression in e.coli (e.coli), the fusion partner is preferably a normal intracellular protein that directs expression to form inclusion bodies. In this case, renaturation of the peptide is not required after cleavage to release the final product. In the present invention, a DNA cassette comprising a fusion partner and a peptide gene may be inserted into an expression vector. Preferably, the expression vector is a plasmid containing an inducible or constitutive promoter to facilitate efficient transcription of the inserted DNA sequence in the host.

The expression vector may be introduced into the host by conventional transformation techniques such as by calcium-mediated techniques, electroporation, or other methods well known to those of ordinary skill in the art.

The sequence encoding the antimicrobial peptide may be derived from a natural source such as a mammalian cell, an existing cDNA or genomic clone, or may be synthetic. One method that can be used is to amplify the antimicrobial peptide by PCR using amplification primers derived from the 5 'and 3' ends of the antimicrobial DNA template and typically incorporating selected restriction sites relative to the cloning site of the vector. Translation initiation and termination codons can be introduced into the primer sequences, if necessary. As long as the codons are selected in consideration of the final mammal to be treated, the sequence encoding the antimicrobial peptide may be codon optimized for expression in a particular host. For example, if the antimicrobial peptide is expressed in bacteria, codon optimization is performed for the bacteria.

The expression vector should contain a promoter sequence to facilitate expression of the introduced antimicrobial peptide. Regulatory sequences such as one or more enhancers, ribosome binding sites, transcription termination signal sequences, secretion signal sequences, origins of replication, selectable markers, and the like may also be included, if necessary. Regulatory sequences are operably linked to each other to allow transcription and subsequent translation. If the antimicrobial peptide is to be expressed in bacteria, the regulatory sequence is one designed for use in bacteria, and is well known to those of ordinary skill in the art. Suitable promoters such as constitutive and inducible promoters are widely available and include those derived from the T5, T7, T3, SP6 phages and the trp, lpp and lac operons.

If the vector containing the antimicrobial peptide is to be expressed in bacteria, examples of origins of replication are those that produce a high copy number or those that produce a low copy number, such as the fl-ori and the col El ori.

Preferably, the plasmid comprises at least one selectable marker functional in the host, which allows the identification and/or selective growth of transformed cells. Suitable selectable marker genes for use in bacterial hosts include ampicillin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, kanamycin resistance gene, and other selectable marker genes known in the art.

Examples of plasmids for expression in bacteria include the pET expression vectors pET3a, pET11a, pET12a-c, and pET15b (available from Novagen, Madison, Wis.). Low copy number vectors (e.g., pPD100) can be used to efficiently overproduce peptides that are detrimental to the E.coli (E.coli) host (Dersch et al, FEMS Microbiol. Lett.123: 19, 1994).

Examples of suitable hosts are bacterial, yeast, insect and mammalian cells. However, bacteria such as e.coli (e.coli) are often used.

The expressed antimicrobial peptides are separated using conventional separation techniques such as affinity, size exclusion or ion exchange chromatography, HPLC, and the like. Different purification techniques can be found in ABiologist's Guide to Principles and techniques of Practical Biochemistry (Wilson and gold, eds., Edward Arnold, London, or Current Protocols in Molecular Biology (John Wiley & Sons, Inc.).

Antimicrobial/pharmaceutical compositions

Furthermore, the present invention relates to an antimicrobial composition/pharmaceutical composition comprising the above antimicrobial peptide and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient. Additional compounds may be included in the composition. These additional compounds include, for example, chelating agents such as EDTA, EGTA or glutathione. Antimicrobial/pharmaceutical compositions having sufficient storage stability and suitable for administration to humans and animals can be prepared by methods known in the art. For example, the pharmaceutical composition may be lyophilized by freeze-drying, spray-drying or spray-cooling.

By "pharmaceutically acceptable" is meant a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient, i.e., the antimicrobial peptide. Such pharmaceutically acceptable buffers, carriers or Excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18 th edition, A.R Gennaro, eds., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3 rd edition, a. kibbe editions, Pharmaceutical Press (2000)).

The term "buffer" means an aqueous solution containing a mixture of acids and bases, the purpose of which is to stabilize the pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, arsenate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolylactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, TES and Tricine.

The term "diluent" means an aqueous or non-aqueous solution, with the purpose of diluting the peptide in the pharmaceutical formulation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or an oil such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil.

The term "adjuvant" means any compound added to a formulation to increase the biological effect of a peptide. The adjuvant may be one or more zinc, copper or silver salts with different anions such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate and acetates with different acyl compositions.

The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition to, for example, facilitate lyophilization.

Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, alginates, carrageenans, hyaluronic acid, polyacrylic acid, polysulfonates, polyethylene glycol/polyethylene oxide, polyvinyl alcohol/polyvinyl acetate with different degrees of hydrolysis and polyvinylpyrrolidone (all of the different molecular weight molecules of these polymers), which are added to the composition, for example for viscosity control, for achieving bioadsorption, or for protecting lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-and triglycerides, ceramides, sphingolipids and glycolipids with different acyl chain lengths and saturations, lecithin of egg, soya lecithin, lecithin of hydrogenated egg and soya lecithin, which are added to the composition for similar reasons as the polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or advantageous coloring properties.

The characteristics of the carrier depend on the route of administration. One route of administration is topical. For example, for topical administration, the preferred carrier is an emulsified cream containing the active peptide, but other commonly used carriers such as some petrolatum/mineral-based and vegetable-based ointments, as well as polymer gels, liquid crystal phases and microemulsions may be used.

The antimicrobial/pharmaceutical composition may comprise one or more peptides, such as 1, 2, 3, or 4 different peptides in the antimicrobial/pharmaceutical composition. By using a combination of different peptides, the antimicrobial effect can be increased and the probability of the microorganism to be combated to develop resistance and/or tolerance against the microbial agent can be reduced.

Peptides, particularly short peptides, based on histidine-rich proteins and/or kininogens have limited antimicrobial activity. However, if the peptides are in a composition comprising salts and/or a pH from about 5.0 to about 7.0, the peptides become active, i.e. an enhanced effect is obtained by the addition of salts and/or the selection of a specific pH range.

The peptide in the form of a salt may be an acid adduct with an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, boric acid, or the like, or an organic acid such as formic acid, acetic acid, haloacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, sulfanilic acid, or the like. Inorganic salts such as monovalent sodium, potassium or divalent zinc, magnesium, copper, calcium, and the like, and corresponding anions, may be added to improve the biological activity of the antimicrobial composition. Antimicrobial H-rich peptides based on kininogen and histidine-rich glycoproteins can be used in defined solutions, such as gels, that determine and control pH (e.g., pH5.5-6.0) to increase the effect of the added antimicrobial peptide. For example, a gel, ointment, or bandage having a defined pH of about 5.0 to about 7.0, such as about 5.5 to about 6.0, with or without ions, will enhance, control, and localize the function of the antimicrobial peptide.

The antimicrobial/pharmaceutical compositions of the present invention may also be in the form of liposomes, wherein the peptides are mixed, in addition to other pharmaceutically acceptable carriers, with amphiphilic agents such as lipids, which are present in aggregated form as micelles, insoluble monolayers and liquid crystals. Suitable lipids for use in liposome formulations include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponins, cholic acids, and the like. The preparation of such liposome formulations can be found, for example, in US4,235,871.

The antimicrobial/pharmaceutical compositions of the present invention may also be in the form of biodegradable microspheres. Aliphatic polyesters such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA), copolymers of PLA and PGA (PLGA), or poly (caprolactone) (PLC) and poly (anhydride) have been widely used as biodegradable polymers in the production of microspheres. The preparation of such microspheres can be found in US5,851,451 and EP 0213303.

Alternatively, the antimicrobial peptide may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or an oil (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil), tragacanth gum and/or various buffers. The pharmaceutical composition may also contain ions and a defined pH to enhance the action of the antimicrobial peptide.

The antimicrobial/pharmaceutical composition may also be subjected to conventional pharmaceutical operations such as sterilization, and/or the composition may contain, for example, conventional adjuvants as disclosed elsewhere herein such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, and the like.

The antimicrobial/pharmaceutical compositions of the present invention may be administered locally or systemically. Routes of administration include topical, ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous and intramuscular), oral, parenteral, vaginal and rectal administration. Administration from implants is also possible. Suitable antimicrobial formulations are, for example, granules, powders, tablets, coated tablet (micro) capsules, suppositories, syrups, emulsions, microemulsions (defined as optically isotropic thermodynamically stable systems consisting of water, oil and surfactant), liquid crystalline phases (defined as systems characterized by long-range order but short-range disorder (e.g. including water-or oil-continuous lamellar, hexagonal and cubic phases)), or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, aerosols, drops or ampoules, and also formulations for the sustained release of the active compounds, where the above-mentioned excipients, diluents, auxiliaries or carriers are conventionally used in these formulations. The pharmaceutical composition of the present invention may also be provided in a bandage, plaster, or the like.

The pharmaceutical composition is administered to a patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose sufficient to produce the desired effect relative to the condition being administered. The exact dosage will depend upon the activity of the compound, the mode of administration, the nature and severity of the disease, the age and weight of the patient and may require different dosages. Administration of a dose may be by one administration in the form of a single dosage unit or several smaller dosage units, and administration of a dose may be by multiple administrations of divided doses at specific intervals.

The pharmaceutical compositions of the present invention may be administered alone or in combination with other therapeutic agents such as antibiotics or antiseptics (antiparasitic agents) such as antibacterial, antifungal, antiviral and antiparasitic agents. Examples are penicillins, cephalosporins, carbacephem, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides and fluoroquinolones. Preservatives include iodine, silver, copper, clorexidine, polyhexamethylene guanidine and other biguanides, chitosan, acetic acid and hydrogen peroxide. These agents may be incorporated as part of the same pharmaceutical composition, or may be administered separately.

The present invention relates to humans and other mammals such as horses, dogs, cats, cattle, pigs, camels, etc. The method is therefore applicable to human therapy and veterinary applications. Targets suitable for such treatment can be identified by established markers of infection such as fever, puls, organism culture, and the like. Infections that may be treated with antimicrobial peptides include infections caused by or due to microorganisms. Examples of microorganisms include bacteria (e.g., gram-positive, gram-negative), fungi (e.g., yeast and mold), parasites (e.g., protozoa, nematodes, cestodes, and trematodes), viruses, and prions. Specific organisms within these classes are well known (see, e.g., Davis et al, Microbiology, 3 rd supplement, Harper & Row, 1980). Infections include, but are not limited to, chronic skin ulcers, infected acute and burn wounds, infected eczema of the skin, impetigo, atopic dermatitis, acne, otitis externa, vaginal infections, seborrheic dermatitis, oral infections and periodontitis, candidal intertrigo, conjunctivitis and other eye infections and pneumonia.

Thus, the antimicrobial/pharmaceutical composition may be used for prophylactic treatment of post-surgical, post-skin injury and burn wounds. The pharmaceutical composition may also be contained in a solution intended for the storage and handling of external substances in contact with the human body, such as contact lenses, orthopedic implants and catheters.

In addition, the antimicrobial/pharmaceutical composition may be used for the treatment of atopic dermatitis, impetigo, chronic skin ulcers, infected acute and burn wounds, acne, otitis externa, fungal infections, pneumonia, seborrheic dermatitis, candidal intertrigo, candidal vaginitis, oropharyngeal candidiasis, eye infections (bacterial conjunctivitis) and nose infections (including with MRSA).

Antimicrobial/pharmaceutical compositions may also be used in lotions, such as lens disinfectants and storage solutions, or may be used to prevent bacterial infections associated with urinary catheter use or central venous catheter use.

Furthermore, the antimicrobial composition may be used to prevent post-operative infections in ointments (plasters), adhesives, sutures or may be incorporated into wound dressings.

Antimicrobial peptides can also be used in polymers, fabrics, etc. to produce antibacterial surfaces or in cosmetic and personal care products (soaps, shampoos, toothpastes, anti-acne agents, sunscreens, tampons, diapers) can be supplemented with antimicrobial/pharmaceutical compositions.

Method for identifying antimicrobial human peptides and/or proteins

The present invention also relates to a method for identifying one or more novel antimicrobial peptides, which allows to provide a mammal, such as a human, with a new set of antimicrobial peptides, which are hypoallergenic and which are effective against microorganisms affecting the mammal. By this means, new and improved antimicrobial peptides will be available, providing a large number of antimicrobial agents, thereby reducing or even eliminating the resistance and/or tolerance problems currently commonly encountered against commercially available antibiotics.

The method comprises the following steps: providing an endogenous peptide and/or protein, providing heparin, mixing the endogenous peptide and/or protein with heparin to produce a peptide and/or protein heparin complex, detecting the peptide and/or protein heparin complex, and identifying an antimicrobial human endogenous peptide and/or protein. In addition, nickel such as nickel sepharose may be used instead of heparin. Heparin may be present in solution, or attached to a substrate. In the latter case, this is suitable for isolation purposes (h.p.l.c or f.p.l.c) or Biocore analysis. For separation purposes, heparin sepharose or similar media may be used. Since antimicrobial peptides also interact with other glycosaminoglycans, these molecules, such as dermatan sulfate or heparan sulfate, can be used for the purification of new antimicrobial peptides. Heparin heparan sulfate and dermatan sulfate contain interspersed and spatially defined sulfo or carboxyl groups. In principle, any other polymeric compound having similar interaction capacity with these glycosaminoglycans may be used for specific binding of antimicrobial peptides. In addition, H-rich peptides can be purified on nickel-sepharose or similar media alone or further in combination with heparin chromatography.

The following examples are intended to illustrate the invention, but are not intended to be limiting in any way, form or manner, either explicitly or implicitly.

Examples

Microorganisms

Enterococcus faecalis (enterococcus faecalis)2374, originally obtained from chronic venous ulcers, escherichia coli (escherichia coli)37.4, Pseudomonas aeruginosa (Pseudomonas aeruginosa)27.4, and Candida albicans (Candida albicans) BM4435 obtained from patients with atopic eczema were used in the experiments.

Example 1

Antimicrobial peptides

The sequence tables and antimicrobial peptides shown in Table 1 below were synthesized by Innovagen AB, Ideon, SE-22370, Lund, Sweden. The purity and molecular weight of these peptides were confirmed by mass spectrometry (maldi. tof Voyager).

TABLE 1

Source	Peptides	Code
			C3a	LRKCCEDGMR ENPMRFSCQR RTRFIS	LRK26
C3a	LGEACKKVFL DCCNYTTELR RQHARAS	LGE27
			C3a	CNYITELRRQHARASHLGLAR	CNY21
Laminin-alpha 1	SRNLSEIKLLISQARK	SRN16
			Laminin-alpha 1	SRNLSEIKLL ISQARKQAAS IKVAVSADR	SRN29
Laminin-alpha 1	KDFLSIELFR GRVKV	KDF15
			Laminin-alpha 1	SAVRKKLSVE LSIRT	SAV15
Laminin-alpha 5	LGTRLRAQSR QRSRPGRWHK VSVRW	LGT25
			Laminin-alpha 5	PPPPLTSASK AIQVFLLGGS RKRVL	PPP25

Laminin-alpha 5	RLRAQSRQRS RPGRWHKVSV RW	RLR22
			Laminin-alpha 1	PGRWHKVSVR.W	PGR11
Laminin-beta 1	RIQNLLKITNLRIKFVKL	RIQ18
			Fibronectin	QPPRARITGYUKYEKPG	QPP18
Von Willebrand factor	YIGLKDRKRP SELRRIASQV KYA	YIG23
			Vitronectin	AKKQRFRHRN RKGYR	AKK15
Protein C inhibitors	SEKTLRKWLK MFKKRQLELY	SEK20
			Histidine-rich glycoproteins	GHHPHGHHPH GHHPHGHHPH	GHH20
Kininogen	KHNLGHGHKH ERDQGHGHQR	KHN20
			Kininogen	GGHVLDHKHGHGHGKHKNKG	GGH20
Kininogen	HKHGHGHGKH KNKGKKNGKH	HKH20
			Synthetic sequences	AKKARAAKKA RAAKKARAAK KARA	AKK24
Synthetic sequences	AKKARAAKKA RAAKKARA	AKK18
			Synthetic sequences	AKKARAAKKA RA	AKK12
Synthetic sequences	ARKKAAKAAR KKAAKAARKK AAKA	ARK24
			Synthetic sequences	ARKKAAKAAR KKAAKA	ARK16
Synthetic K->H sequence	AHHAHAAHHA HAAHHAHAAH HAHA	AHH24:1
			Synthetic K->H sequence	AHHHAAHAA HHHAAHAAHHH AAHA	AHH24:2

Example 2

Antibacterial effect of arginine and lysine-rich peptides

FIG. 1 depicts the bactericidal effect of arginine and lysine rich peptides (sequence listing) on Enterococcus faecalis (Enterococcus faecalis). Bacteria were cultured in Todd-Hewitt (TH) medium to mid-log phase. The bacteria were washed and diluted with 10mM Tris, pH7.4, containing 5mM glucose. Incubation of bacteria with synthetic peptide at 37 deg.C (50. mu.l; 2X 10)⁶cfu/ml) for 2 hours, wherein the synthetic peptide is at a concentration of 0.03 to 60. mu.M. To quantify the bactericidal activity, serial dilutions of the incubation mixture were plated on TH agar plates, followed by overnight incubation at 37 ℃, and the number of colony forming units was determined.

2x10⁶Colony Forming Unit (CFU). times.ml^-1Enterococcus faecalis (e.faecalis) (isolate 2374) was incubated with peptide at a concentration of 0.03 to 60 μ M in 50 μ l. (A) A synthetic peptide derived from laminin. The sequences derived from the alpha 5 chain (PPP 25: SEQ ID NO: 13, LGT 25: SEQ ID NO: 12, RLR 22: SEQ ID NO: 14, PGR 11: SEQ ID NO: 15) and the alpha 1 chain (SRN 16: SEQ ID NO: 8, SRN 29: SEQ ID NO: 9, KDF 15: SEQ ID NO: 10, SAV 15:SEQ ID NO: 11) the effect of the peptide of LG domain of (1). One peptide (RIQ 18: SEQ ID NO: 16) was derived from the beta 1 chain. (B) Three peptides were derived from complement factor C3(LRK 26: SEQ ID NO: 5, LGE 27: SEQ ID NO: 6 and CNY 21: SEQ ID NO: 7), AKK15 was derived from vitronectin, SEK 20: SEQ ID NO: 19 is derived from a protein C inhibitor, QPP 18: SEQ ID NO: 17 was derived from fibronectin and YIG 23: SEQ ID NO: 18 is derived from von Willebrand factor. (C) Heparin binding consensus sequence (AKKARA)_n(n-1-4) and (ARKKAAKA)_n(n-1-3) antibacterial effect. The n-1 peptide does not exert an antimicrobial effect. Peptides that do not interact with heparin: GHRPLDKKREEAPSLRPA, LVTSKGDKELRTGKEKVTS and KNNQKSEPLIGRKKT (Andersson et al, EurJBiochem, 2004, 271; 271: 1219-.

Example 3

Radial diffusion assay analysis of antimicrobial peptides (Table 2)

Radial Diffusion Assays (RDA) were performed essentially as previously described (Andersson et al, Eur J Biochem, 2004, 271: 1219-. Briefly, bacteria (e.coli) or fungi (candida albicans) were cultured in 10ml of full strength (3% w/v) Tryptic Soy Broth (TSB) (Becton-Dickinson, cockaysville, MD) to mid-log phase. The microorganisms were washed 1 time with 10mM Tris, pH 7.4. To 5ml of a lower agarose gel consisting of 0.03% (w/v) TSB, 1% (w/v) Low electroosmosis (Low-EEO) agarose (Sigma, St Louise MO) and a final concentration of 0.02% (v/v) Tween20(Sigma) was added 4X10⁶Bacteria cfu or 1x10⁵Fungi cfu. To the direction ofThe lower agarose gel was poured into an 85mm petri dish. After agarose solidification, 4mm diameter wells were punched out and 6 μ l of the sample to be tested was added to each well. The plates were incubated at 37 ℃ for 3 hours to allow the peptide to diffuse. Then 5ml of melted cover layer (dH) was used₂6% TSB in O and 1% Low-EEO agarose) was overlaid on the lower gel. After incubation at 37 ℃ for 18-24 hours, the antimicrobial activity of the peptides was observed through the clear zone surrounding each well. Assay for combinations of 100. mu.M concentrationsPeptide formation to determine antibacterial effect relative to the known peptide LL-37. To minimize the differences between the experiments, LL-37 standard (100. mu.M) was included on each plate. The activity of the peptide is expressed in radial diffusion units (diameter of the clearing zone in mm-pore diameter) × 10). The results are shown in Table 2 below.

TABLE 2

Source	Code	Radial diffusion unit
			hCAP-18	LL-37	50
C3a	LRK26	70
			C3a	LGE27	40
C3a	CNY21	32
			Laminin-alpha 1	SRN16	77
Laminin-alpha 1	SRN29	71

Laminin-alpha 1	KDF15	65
			Laminin-alpha 1	SAV15	75
Laminin-alpha 5	LGT25	85
			Laminin-alpha 5	PPP25	81
Laminin-alpha 5	RLR22	92
			Laminin-alpha 1	PGR11	86
Laminin-beta 1	RIQ18	93
			Fibronectin	QPP18	59
Von Willebrand factor	YIG23	80
			Vitronectin	AKK15	101
Protein C inhibitors	SEK20	92
			Synthetic sequences	AKK24	67
Synthetic sequences	ARK24	74

Example 4

Radial diffusion assay of peptides against E.coli (E.coli) and Candida albicans (C.albicans) (FIG. 2)

Figure 2 shows a radial diffusion assay using a panel of antimicrobial peptides. The assay was performed as described above. Antimicrobial activity of the peptides was observed through the clear zone around each well after incubation of enterococcus faecalis (e.faecalis) bacteria at 37 ℃ (panel a) and Candida albicans (Candida albicans) at 28 ℃ (panel B) for 18-24 hours.

Example 5

Antibacterial effect of histidine-rich peptides

FIG. 3 depicts the bactericidal effect of histidine-rich peptides. Enterococcus faecalis (e.faecalis) bacteria were cultured to mid-log phase in Todd-hewitt (th) medium. The bacteria were washed and diluted in 10mM Tris, pH7.4 with 5mM glucose, with or without 50. mu.M ZnCl, or in 10mM MES buffer, 5mM glucose, pH 5.5. Incubate bacteria at 37 deg.C (50. mu.l; 2X 10)⁶cfu/ml) and synthetic peptide at a concentration of 0.03 to 60. mu.M (Tris buffer, with or without zinc) or 30 and 60. mu.M (Tris and MES buffers) for 2 hours. To quantify the bactericidal activity, serial dilutions of the incubation mixture were plated on TH agar plates, followed by overnight incubation at 37 ℃, and the number of colony forming units was determined. (A) Shows the effect of peptides derived from the heparin-binding domain of histidine-rich glycoprotein (GHH 20: SEQ ID NO: 4) and kininogen (KHN 20: SEQ ID NO: 3, GGH 20: SEQ ID NO: 2 and HKHH 20: SEQ ID NO: 1) with or without 50 μ M ZnCl. (B) The method comprises the following steps Action of the peptide (30 and 60. mu.M) in 10mM Tris, pH7.4 with 5mM glucose or in 10mM MES buffer, 5mM glucose, pH 5.5. Numbers indicate% survival, where 100% is control (no peptide). (C) The method comprises the following steps Effect of peptides AHH24:1 and AHH24:2 on enterococcus faecalis (e.faecalis) with fixed peptide/zinc molar ratio (1: 100). The peptide does not exert antimicrobial activity in the absence of zinc.

Example 6

Electron microscopy analysis of peptide effects

FIG. 4 shows electron microscopy analysis of Pseudomonas aeruginosa (Pseudomonas aeruginosa) bacteria subjected to the action of antimicrobial peptides. (A) The method comprises the following steps And (6) comparison. (B-H) analysis of bacteria treated with peptide at about 50% of the desired bactericidal concentration. 200% of HKH20 was also analyzed. (B) LL-37, (C) ARK24, (D) SEK20, (E) AKK24, (F) LGT25, (G) HKH20 and (H) HKH20 at 200% bactericidal concentration. The bars represent 1 μm, except for G and H (0.5 μm). Electron microscopy analysis of the peptide-treated bacteria demonstrated that the treated bacteria were morphologically distinct compared to the control. Cathelicidin LL-37 caused local perturbation and disruption along the bacterial cell membrane of pseudomonas aeruginosa (p. aeruginosa) and occasionally intracellular material was found extracellularly, and similar observations were obtained with the endogenous antimicrobial peptides disclosed herein.

Example 7

Heparin binding of endogenous antimicrobial peptides (FIG. 5)

The peptides were tested for heparin binding activity. Peptides were applied on nitrocellulose membranes (Hybond, Amersham Biosciences). The membranes (PBS, ph7.4, 0.25% Tween20, 3% bovine serum albumin) were blocked for 1 hour and incubated with radiolabeled heparin in the same buffer for 1 hour. Heparin binding of histidine-rich peptides was detected with or without 50 μ M ZnCl. Radioiodination of heparin was performed as described previously (Andersson et al, Eur JBiochem, 2004, 271; 271: 1219-. Unlabeled polysaccharide (2mg/ml) was added for competitive binding. Wash membranes (PBS, pH7.4, 3X10min in 0.25% Tween 20). Radioactivity was observed using a Bas2000 radiological imaging system (Fuji).

Unlabeled heparin (6mg/ml) inhibition¹²⁵Binding of I-heparin to C3-derived peptides LRK26 and LGE27 and LL-37 (upper panel).

Example 8

Purification of histidine-containing antimicrobial fragments on a Nickel-agarose gel (FIG. 6)

Domain D5 of human kininogen, which contains the peptide epitopes KHN20, GGH20 and HKH20, is expressed in the e.coli (e.coli) strain (BL21DE 3). Protein production was induced by the addition of 1mM isopropyl-thio- β -D-galactoside to exponentially growing bacteria. After 3h incubation, the bacteria were collected by centrifugation. The pellet was resuspended in 50mM phosphate, 300mM NaCl, pH8.0 (buffer A) and the bacteria lysed by repeated freeze-thaw cycles. The lysate was then centrifuged at 29000g for 30 minutes. The supernatant was mixed with 2ml NiNTA-agarose loaded with nickel and equilibrated with buffer A. Agarose was applied to the column and washed with 10ml of buffer A containing 0.1% Triton X-100, 10ml of buffer A, 5ml of buffer A containing 1M NaCl, 5ml of buffer A, 10ml of 20% ethanol, 10ml of buffer A containing 5mM imidazole and buffer A containing 30mM imidazole. The protein was eluted in 500mM imidazole (arrow). This domain exerts an antibacterial effect against e.coli (e.coli) in a radial diffusion assay.

Sequence listing

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<120> novel antimicrobial peptides having heparin-binding activity

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Claims (12)

1. Use of a peptide having heparin binding activity, derived from C3a, and having from 10 to 36 amino acid residues, wherein said peptide comprises at least 1 amino acid sequence selected from the group consisting of SEQ ID NOs 5,6, or 7, for the preparation of an antimicrobial composition.

2. Use according to claim 1, wherein the peptide comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acid residues.

3. Use according to claim 1, wherein the peptide is endogenous, synthetic or semi-synthetic.

4. Use according to claim 1, wherein the peptide is modified by substitution of 1-6 amino acids.

5. Use according to claim 1, wherein the peptide is modified by amidation, esterification, acylation, acetylation, PEGylation or alkylation.

6. Use according to claim 1, wherein said composition is a pharmaceutical composition comprising said peptide and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.

7. Use according to claim 1, wherein the composition is in the form of granules, powders, tablets, coated tablets, capsules, suppositories, syrups, emulsions, gels, ointments, suspensions, creams, aerosols, drops or injectable forms.

8. Use according to claim 1, wherein the antimicrobial composition is for preventing, inhibiting, reducing or destroying a microorganism selected from the group consisting of bacteria, viruses, parasites, fungi and yeasts.

9. Use according to claim 1, wherein the antimicrobial composition is used in therapy or diagnosis.

10. Use according to claim 1, wherein the antimicrobial composition is for the treatment of antimicrobial diseases caused by microorganisms selected from the group consisting of bacteria, viruses, parasites, fungi and yeasts.

11. Use according to claim 10, wherein the antimicrobial composition is for the treatment of a disease caused by a microorganism selected from the group consisting of Enterococcus faecalis (Enterococcus faecalis), escherichia coli (escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Proteus mirabilis (Proteus mirabilis), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus pyogenes (Streptococcus pyogenes), and Staphylococcus aureus (Staphylococcus aureus).

12. Use according to claim 10, wherein the antimicrobial composition is for the treatment of a disease caused by a microorganism selected from the group consisting of Candida albicans (Candida albicans) and Candida parapsilosis (Candida parapsilosis).