WO2011115562A1 - Antimicrobial compound - Google Patents

Abstract

The invention relates to a peptide having a length of from 10 to 40 amino acid residues being derived from either SEQ IDNO:1 or SEQ ID NO:4 or which has at least 75 % homology to SEQ ID NO:1 or 4 or wherein SEQ IDNO:1or SEQ ID NO:4 has one or more substitutions and being active against bacteria or fungus or mixtures thereof, a pharmaceutical composition comprising said peptide, use of said peptide for the manufacturing of a medicament for the treatment of a bacterial of fungal disease or a mixture thereof as well a method of treatment of a mammal suffering from mentioned disease.

Description

ANTIMICROBIAL COMPOUND

FIELD OF INVENTION

The invention relates to a peptide having a length of from 10 to 40 amino acid residues being derived from either SEQ IDNO: l or SEQ ID NO:4 or which has at least 75 % homology to SEQ ID NO: 1 or 4 or has one or more substitutions and being active against bacteria or fungus or mixtures thereof, a pharmaceutical composition comprising said peptide, use of said peptide for the manufacturing of a medicament for the treatment of a bacterial of fungal disease or a mixture thereof as well a method of treatment of a mammal suffering from mentioned disease.

BACKGROUND OF THE INVENTION

Drug resistant micro-organisms, especially bacteria, are becoming increasingly problematic as infection rates continue to rise and effective methods of control become more and more limited. Prolific use of antibiotics over the last 50 or so years together with the indiscriminate prescribing of antibiotics and patient noncompliance with treatment regimes, has selected for micro-organisms that have developed or acquired ways of overcoming the effects of antibiotics. The

transmission and control of drug-resistant organisms is becoming one of the most significant problems within healthcare. Therefore, it is of interest to develop new antimicrobial compounds.lt is known that the human body can produce peptide antibiotics to combat bacterial and fungal infections (Hancock & Sahl, 2006).

Examples of such antimicrobial peptides are cathelicidins and defensins. At present, over 700 different antimicrobial peptide sequences are known (See for example www.bbcm.univ.trieste.it/~tossi/search.htm; http://aps.unmc.edu/AP/main.php).

Midkine (MK) and Pleiotrophin (PTN) constitute a family of developmentally regulated secreted heparin-binding proteins now known as the Neurite Growth-promoting Factor (NEGF) family (Laaroubi et al, 1995). MK is also called Retinoic Acid-Inducible Heparin Binding Protein (RIHB) or Neurite Growth-Promoting Factor 2 (NEGF-2). It is a 13.4 kDa polypeptide, containing 123 amino acid residues, including five intra-molecular disulfide bonds. PTN is also called Neurite Growth-Promoting Factor 1 (NEGF-1), Heparin Binding Growth- Associated Molecule (HB-GAM), OSF-1, HARP, or HBNF. Pleiotrophin is a 15.4 kDa polypeptide, containing 136 amino acid residues, and five intra-molecular disulfide bonds (Muramatsu, 2002).

Both MK and PTN have a high content of cationic amino acids (pi of 10.0 and 10.2 respectively) and cysteines. The molecules are 45% homologous with each other on the amino acid level. MK and PTN bind and activate the same receptor, known as Anaplastic Lymphoma Kinase (ALK), which is expressed on many, phenotypically different, cells. The expression pattern of MK and PTN during embryogenesis suggests that they have important roles in neurogenesis, cell migration, secondary organogenetic induction, and mesoderm epithelial interaction (Muramatsu, 2002). Recently, it was demonstrated that the MK gene has a NF-kappaB responsive element in its promoter-region rendering the gene responsive to pro-inflammatory stimuli (You et al, 2008). SUMMARY OF THE INVENTION

The invention relates to a peptide having a length of from 10 to 40 amino acid residues being derived from either SEQ IDNO: l or SEQ ID NO:4 or which has at least 75 % homology to SEQ ID NO: 1 or 4 or wherein SEQ ID NO: 1 or SEQ ID NO:4 has one or more substitutions and being active against bacteria or fungus or yeasts or mixtures thereof. The invented peptides show an increased activity against bacteria as well as fungi compared to other peptide which are known to the inventors.

Additionally, the invented peptides are more stable and more interestingly shows stability in a saline environment which has so far been unknown for other peptides that are derived from mammalian proteins. The stability to salt gives rise to the possibility to use the peptides not only to treat dermatological diseases, caused by fungi as well as bacteria. The invented peptides may as well be used to prevent an infection in a mammal such as a human being. An infection caused by bacteria and fungi or mixtures thereof. It may as well be used prior to surgery.

Furthermore, the present invention relates to a pharmaceutical composition which comprises the peptide defined above and a pharmaceutical acceptable adjuvant, carrier, buffer or diluent. The present invention further relates to a pharmaceutical composition which comprises at least one peptide as defined above and a pharmaceutical acceptable adjuvant, carrier, buffer or diluent.

Additionally, the invention relates to the use of the peptide defined above for the manufacturing of a medicament for the treatment or prevention of an infection caused by bacteria or fungi or a mixture thereof.

Finally, the invention relates to and a method of treating an infection comprising administering a pharmaceutical composition as defined above to a mammal suffering from an infection caused by bacteria, fungi or a mixture thereof.

The new invented peptides will reduce the risk for allergic reactions to antimicrobial peptides due to the fact that the peptides are derived from the polypeptide sequence of endogenous proteins and/or peptides. By using short peptides the stability of the peptide is increased and the production costs reduced, as compared to longer peptides and proteins, whereby the invention may be

economically advantageous.

Furthermore, the use of these new peptides with specific or preferable actions on Gram-negative and Gram-positive bacteria, or fungi, enables specific targeting of various microorganisms, thus minimising development of resistance and ecological problems. By using supplementing peptides, which are comparable to peptides already existing in the mammal, the risk of additional ecological pressure by novel antibiotics is further diminished.

The inventive antimicrobial peptides increase the list of antimicrobial agents, which aid in the choice to prevent, reduce or eliminate microorganisms in all kind of applications including but not limited to those that invade or infect mammals, such as the human being. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Bactericidal activity of human midkine and pleiotrophin. A - D, Midkine (MK) and pleiotrophin (PTN) were investigated for antibacterial activity using viable count assay. The gram-positive pathogens Streptococcus pyogenes (S. pyogenes) and Staphylococcus aureus (S. aureus), and the gram- negative pathogens Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), were grown to mid- logarithmic phase followed by incubation with polypeptides at the indicated concentrations or buffer alone, for one hour at 37°C. Serial dilutions were made and after culture overnight on agar plates, the number of colony forming units (CFU) was counted. The number of colonies remaining after exposure to MK and PTN respectively was compared with the number of CFU obtained after incubation in buffer alone, to calculate % killing. The data shown represent mean, ±standard deviation from three separate experiments.

Figure 2. Antibacterial activity of MK and PTN in the presence of salt and plasma. To investigate if the presence of sodium chloride and plasma proteins interfere with the bactericidal activity of MK and PTN respectively, the viable count assay was used. S. aureus and E. coli were grown to mid-logarithmic phase followed by incubation with MK and PTN, respectively (0.5 μΜ in the case of S. aureus and 1 μΜ in the case of E. coli) for one hour, in the absence or presence of sodium chloride at the indicated concentration (A and B) or a combination of sodium chloride (150 mM) and plasma at the indicated concentration (C and D). After incubation, serial dilutions were plated and the resulting CFU counted. The data shown represents mean, ±standard deviation from three separate experiments.

Figure 3. Membrane-disrupting properties of human MK and PTN. A and B, Membrane-disruptive effects of MK (A) and PTN (B) were analyzed by measuring release of carboxyfluorescein from liposomes in the absence or presence of sodium chloride (150 mM). A dose-dependent leakage, that was not affected by the presence of sodium chloride, was seen. C, Ultrastructural changes of bacterial morphology after incubation with MK or PTN was investigated using electron microscopy. S. pyogenes and E. coli were incubated in buffer alone (control), or in the presence of MK (at 0.5 and 1 μΜ respectively), and with PTN (0.5 μΜ in the case of S. pyogenes and 1 μΜ in the case of E. coli), respectively. The samples were then fixed and processed for negative staining. The electron micrographs show intact bacteria after incubation in buffer alone (control) while incubation in the presence of MK and PTN caused bacterial disintegration with membrane blebbing and leakage of intracellular contents. Bar=0.5 μηι

Figure 4: Fungicidal activity of MK and PTN. A-B. Using a viable count assay the fungicidal activity of MK and PTN were investigated. Candida albicans (A) and Candida parapsilosis (B) were grown to mid-logarithmic phase and incubated with MK or PTN (concentrations ranging from 0.01-3μΜ) or in buffer alone, for one hour at 28°C. Serial dilutions were made and after culture overnight on agar plates the number of colony forming units (cfu) were counted and compared to the control. The data shown represent mean, ±standard deviation from three separate

experiments.

Figure 5: Fungicidal activity of human MK and PTN in the presence of salt. To determine whether the sodium chloride interfere with the fungicidal activity of MK and PTN the viable count assay with addition of sodium chloride (50-150mM) was used. C. albicans (A, B) and C. parapsilosis (C, D) were grown to mid-logarithmic phase followed by incubation with MK (A, C) and PTN (B, D) (3μΜ) or in buffer alone, for one hour at 28°C. Serial dilutions were plated and the resulting cfu counted. The data shown represents mean, ±standard deviation from three separate experiments.

Figure 6: A. Radial diffusion assay was performed on MK-peptides. C. albicans were grown to midlogaritmic phase, added to Petri dishes with agarose and allowed to solidify. Wells were punched and peptide, or buffer alone, was added. Plates were incubated at 28°C for 3 h to allow the peptides to diffuse. Antifungal activity was seen as a clearing zone around each well after incubation for 18-24h at 28°C. B. Overlapping peptides, 20 amino acids in length, were derived from the human MK sequences. Figure 7: Fungicidal regions of MK. A. The four most fungicidal peptides, 20 amino acids in length and overlapping, derived from the human MK sequence were subjected to viable count assay at concentrations ranging from l-ΙΟΟμΜ. C.

albicans were grown to mid-logarithmic phase followed by incubation with MK- peptides (MK5, MK9, MK1 1, MK12) in the indicated concentrations or in buffer alone, for one hour at 28 °C.

B-E. The selected MK-peptides (30 μΜ) were also incubated in the presence of physiological concentrations of sodium chloride using viable count assay (B: MK5; C: MK9; D: MK1 1 ; E: MK12). The data shown represents mean, ±standard deviation from three separate experiments.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "antimicrobial polypeptide of the invention" is intended to include SEQ ID NO: l and SEQ ID NO:4 as such, fragments thereof, such as SEQ ID NO:2-3 and 5-31 as well as modified whole polypeptide and fragments of the modified polypeptides.

The term "antimicrobial" is intended to mean an activity, which prevents, inhibits, reduces or destroys a microorganism. The antimicrobial activity can be determined by any method, such as the method in EXAMPLE 1. In analogy, the term "anti-bacterial" is intended to mean an activity which prevents, inhibits, reduces or destroys bacteria; the term "anti-fungal" is intended to mean an activity, which prevents, inhibits, reduces or destroys fungus or yeast; the term "anti- parasitic" is intended to mean an activity, which prevents, inhibits, reduces or destroys parasites.

In this context the terms anti-fungal and fungicidal are used interchangeably.

The term "antimicrobial agent" is intended to mean any agent, which prevent, inhibit or destroy life of microorganisms. Examples of antimicrobial agents can be found in The Sanford Guide to Antimicrobial Therapy (32nd edition, Antimicrobial Therapy, Inc, US).

The term "microorganism" is intended to mean any living microorganism. Examples of microorganisms are bacteria, fungi and yeasts.

The term "homologue thereof is intended to mean that part of or the whole of a polypeptide is related to another polypeptide. See further "homology".

The term "homology" is intended to mean the overall homology to the polypeptide (SEQ ID NO: l or SEQ ID NO:2). The degree of homology is an indication of the degree of relatedness between two sequences. Homology is commonly examined by means of alignments, using computer-based tools.

Examples of such tools are BLAST (found at

http://www.ncbi.nlm.nih.gov/BLAST/) and FASTA (found at

http://www.ebi.ac.uk/Tools/fasta/) .The degree of homology may be further specified by indicating percentage similarity or percentage identity. The term

"identity" in this context is intended to indicate residues that are identical in the two compared sequences. The term "similarity" in this context is intended to indicate to mean how many residues are similar, i.e. belong to the same group (e.g.

hydrophobic, hydrophilic).

The term "carrier" is intended to mean any type of molecule that is directly or indirectly associated or linked with the polypeptide of the invention.

The term "linked" is intended to mean "linked" with covalent or chemical bonds.

In the present context, amino acid names and atom names are used as defined by the Protein DataBank (PNB) (www.pdb.org), which is based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom names etc.), Eur J Biochem., 138, 9-37 (1984) together with their corrections in Eur J Biochem., 152, 1 (1985). The term "amino acid" is intended to indicate an amino acid from the group consisting of 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 (lie 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 ), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y), or derivatives thereof.

The term "substituted" is intended to mean that an amino acid residue is replaced by another amino acid residue. For example, S19V means that the serine amino acid residue in position number 19 in SEQ ID NO: l has been substituted, i.e., replaced by valine.

Peptides of the invention

The invention relates to peptides having a length of from 10 to 40 amino acid residues being derived from either SEQ IDNO: l or SEQ ID NO:4 or which has at least 75 % homology to SEQ ID NO: 1 or 4 or wherein SEQ ID NO: 1 or SEQ ID NO:4 has one or more substitutions and being active against bacteria or fungus or mixtures thereof.

The peptides of the invention may show for example about 75% similarity, over 75%, over 80%, over 85%, over 90%, over 95%, over 96%, over 97%, over 98% or over 99% similarity with SEQ ID NO: l or SEQ ID NO:4. In a further embodiment the peptides of the invention may have an identity to SEQ ID NO: l or SEQ ID NO:4 of about 75%, about 80%, about 85%, about 90%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%.

In a further embodiment the peptides of the invention relates to a peptide as defined above, wherein said peptide is SEQ ID NO:2-3 or 5-31 or which has at least 75 % homology to SEQ ID NO:2-3 or 5-31 or wherein SEQ ID NO:2-3 or 5-31 has one or more substitutions.

In a further embodiment the peptides of the invention relate to peptide wherein said peptide is SEQ ID NO.2 (MK5), SEQ ID NO.15 (MK9), SEQ ID NO. 17 (MK1 1), SEQ ID NO. 3 (MK 12), SEQ ID NO.5 (PTN 5) or SEQ ID NO. 6 (PTN 12).

The peptides of the invention may contain 1 , 2,3 4, 5 or up to 6 substitutions and having a length of from 10-30, 15-30, 15-25 or 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27 ,28 ,29,30, 31, 32, 33, 34,35, 36, 37, 38 or 29 amino acid residues, such as 20-25 amino acid residues. The length of the peptide being dependent on the amino acid residues of the peptide as well as the micororganism to combat or the stability of the peptide. However, for economical reasons smaller peptides are preferred.

Additionally, the invented peptides are found to be active in salt (see the examples below). This means that the invented peptides are useful to be used to combat infections inside the body of a mammal, such as a human being or other animal, such as cow, horse, cat, dog, camel etc. Examples of diseases that the salt stable peptides may be used against includes impetigo, erysipelas, bacterial and fungal wound infections, bacterial and fungal prosthesis infections, bacterial and fungal arthritis, bacterial and fungal conjunctivitis, bacterial and fungal pharyngitis, bacterial and fungal laryngitis, bacterial and fungal acute bronchitis, bacterial and fungal exacerbations in COPD and cystic fibrosis, bacterial and fungal pneumonia, bacterial and fungal gastritis, bacterial and fungal gastroenteritis, bacterial and fungal colitis, bacterial and fungal meningitis, bacterial and fungal cerebral abscess, bacterial and fungal external otitis, bacterial and fungal otitis media, bacterial and fungal osteomyelitis, bacterial and fungal pleural empyema, parodontitis, bacterial and fungal vulvitis, bacterial and fungal vaginitis, bacterial and fungal salpingitis, bacterial and fungal peritonitis, bacterial and fungal rhinitis, bacterial and fungal mucositis, bacterial and fungal urethritis, bacterial and fungal epididymitis, bacterial and fungal prostatitis, bacterial and fungal endocarditis, necrotizing fasciitis, and bacterial and fungal septicemia, infected acute wounds, chronic wounds, topical wounds, burn wounds including sunburn and thermal burns, ulcers such as chronic skin ulcers or stomach ulcers, paronychia, microbial keratitis, mastitis, acne,impetigo, infected skin eczema, atopic dermatitis, vaginal infections, external otitis, seborrhoic dermatitis, oral infections and parodontitis, candidal intertrigo, conjunctivitis and other eye infections such as P. aeruginosa keratitis; pneumonia and other airway diseases; and gastrointestinal disorders. In addition, diseases caused by viruses (e.g. HIV, Herpes simplex, Papilloma- viruses, and rhinoviruses).

In a further embodiment the invention relates to a peptide as defined above, wherein said peptide is SEQ ID NO:2-3 or 5-31 or which has at least 75 % homology to SEQ ID NO:2-3 or 5-31 or wherein SEQ ID NO:2-3 or 5-31 has one or more substitutions. Those sequences are derived from SEQ ID NO: l and 2.

Those sequences may also be derived from SEQ ID NO: 1 and 4.

The peptides shown in SEQ ID NO:2-3 or 5 to 31 shows even a higher activity against bacteria and fungi compared to the other peptides mentioned above, but all of the peptides may be of interest due to that all shows interesting activity against the different microorganisms and it might be that during the development of the peptides into a pharmaceutical formulations the most active ones are less interesting compared to the other ones due to for example, stability, price to produce, toxicity etc.

The peptides of the invention may be prepared from different sources, for example from natural sources by means of purification from eukaryotic cells naturally expressing the molecules in question, or by recombinant means or synthetically. Examples of recombinant means are in vitro translation of mRNA obtained from cells naturally expressing the molecules in question, by chemical synthesis or by expression and purification from transfected cells of mammalian, yeast, bacterial or other origin.

The peptides of the invention may also be prepared synthetically. For example, the synthesis of the peptides may be performed according to standard chemical methods, including synthesis by automated procedure. In general, peptide analogues are synthesised based on the standard solid-phase Fmoc protection strategy with HATU (N- [DIMETHYL AMINO - 1 H- 1.2.3. -TRI AZOLO [4,5- BJPYRIDIN- 1 -YLMETHYLELEJ-N-METHYLMETHANAMINIUM

HEXAFLUOROPHOS-PHATE N-OXIDE) as the coupling agent or other coupling agents such as HO At- 1 -HYDROXY-7-AZABENZOTRIAZOLE. The peptide is cleaved from the solid-phase resin with trifluoroacetic acid containing appropriate scavengers, which also deprotects side chain functional groups. Crude peptide is further purified using preparative reversed-phase chromatography. Other

purification methods, such as partition chromatography, gel filtration, gel electrophoresis, or ion-exchange chro-matography may be used. Other synthesis techniques, known in the art, such as the tBoc protection strategy, or use of different coupling reagents or the like can be em-ployed to produce equivalent peptides. The peptide the invention may alternatively be synthesised by recombinant production (see e.g., U.S. Pat. No. 5,593,866). A variety of host systems are suitable for production of the polypeptides of the invention, including bacteria, such as E. coli, yeast, such as Saccharomyces cerevisiae or pichia, insects, such as Sf9, and mammalian cells, such as CHO or COS-7. There are many expression vectors available to be used for each of the hosts and the invention is not limited to any of them as long as the vector and host are able to produce the antimicrobial

polypeptide. Vectors and procedures for cloning and expression in E. coli can be found in for example Sambrook et al. (Molecular Cloning.: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1987) and

Ausubel et al. (Current Protocols in Molecular Biology, Greene Publishing Co., 1995).

One example of a method for synthesising a recombinant peptide of the invention is as follows: A DNA sequence encoding the peptide may be introduced into a suitable expression vector appropriate for the host. In one preferred

embodiment, the gene is cloned into a vector to create a fusion protein. To facilitate isolation of the polypeptide sequence, amino acids susceptible to chemical cleavage (e.g., CNBr) or enzymatic cleavage (e.g., V8 protease, trypsin) may be used to bridge the peptide and fusion partner. For expression in E. coli, the fusion partner is preferably a normal intracellular protein that directs expression toward inclusion body formation. In such a case, following cleavage to release the final product, there is no requirement for renaturation of the peptide. In one embodiment, the DNA cassette, comprising fusion partner and peptide gene, may be inserted into an expression vector. Preferably, the expression vector is a plasmid that contains an inducible or constitutive promoter to facilitate the efficient transcription of the inserted DNA sequence in the host.

The expression vector can be introduced into the host by conventional trans-formation techniques such as by calcium -mediated techniques,

electroporation, or other methods well known to those skilled in the art.

The sequence encoding the peptide may be derived optionally from a natural source such as a mammalian cell, or an existing cDNA or genomic clone or be synthesised. An example of one method which may be used, is the amplification of the sequence of the peptide by the aid of PC using amplification primers which are derived from the 5' and 3' ends of the DNA template and typically incorporate restriction sites chosen with regard to the cloning site of the vector. If necessary, translational initiation and termination codons may be engineered into the primer sequences. The sequence encoding the peptide may be codon-optimised to facilitate expression in the particular host as long as the choice of the codons is made considering the final mammal to be treated. Thus, for example, if the peptide is expressed in bacteria, the codons are optimised for bacteria.

The expression vector may contain a promoter sequence, to facilitate expression of the introduced peptide. If necessary, regulatory sequences may also be included, such as one or more enhancers, ribosome binding site, transcription termination signal sequence, secretion signal sequence, origin of replication, selectable marker, and the like. The regulatory sequences may be operably linked to each other to allow transcription and subsequent translation. If the antimicrobial polypeptide is to be expressed in bacteria, the regulatory sequences are those which are designed to be used within bacteria and such are well-known for a person skilled in the art. Suitable promoters, such as constitutive and inducible promoters, are widely available and include promoters from T5, T7, T3, SP6 phages, and the trp, lpp, and lac operons.

If the vector containing the peptide is to be expressed within bacteria examples of origin are either those, which give rise to a high copy number or those which give rise to a low copy, for example fl-ori and col El ori.

Preferably, the plasmids include at least one selectable marker that is functional in the host, which allows transformed cells to be identified and/or selectively grown. Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, kanamycin resistance gene and others known in the art.

Examples of plasmids for expression in bacteria include the pET expression vectors pET3a, pET 1 la, pET 12a-c, and pET 15b (available from Novagen, Madi-son, Wis.). Low copy number vectors (e.g., pPDIOO) can be used for efficient over- production of peptides deleterious to the E. coli host (Dersch et al, FEMS Microbiol. Lett. 123: 19, 1994).

Examples of suitable hosts are bacteria, yeast, insects and mammal cells. However, often bacteria such as for example E.coli, is used.

The expressed peptide may be isolated by conventional isolation techniques such as affinity, size exclusion, ionic exchange chromatography, or HPLC and the like. Different purification techniques can be found in A Biologist^'s Guide to Principles and Techniques of Practical Biochemistry (eds. Wilson and Golding, Edward Arnold, London), or in Current Protocols in Molecular Biology (John Wiley & Sons, Inc).

The genetic material used for transfection/transformation may for example be of natural origin isolated from cells, tissue or organisms. Alternatively, it may be of synthetic origin e.g. synthetic genes identical to the natural DNA sequence, cDNA, genomic clone, chemically synthesises or obtained by recombinant DNA techniques. The DNA may furthermore be modified to introduce molecular changes or to ease recombinant expression.

Finally, the peptides may be purified from plasma, blood, various tissues or the like. The peptides may be endogenous or generated after enzymatic or chemical digestion of the purified protein. For example, a protein may be digested by trypsin and the resulting peptides further isolated in larger scale.

The present invention also comprises modified variants of the peptides defined above, such as substitutions. Such variants of peptides which are useful in antimicrobial preparations and compositions are also included in this invention. Modifications may be aimed to influence for example the solubility of the peptide, resistance to protease degradation, uptake into cells, penetration, efficacy, ease of purification, amounts produced.

The modified peptides of the invention may be modified at nucleotide sequence level. For example, the genetic material may contain modified or added designer domain(s) or sequence(s). One example is such as to include promoter regions which give increased secretion of the instant peptide or peptides. If necessary, regulatory sequences may also be included, such as one or more enhancers, ribosome binding site, transcription termination signal sequence, secretion signal sequence, origin of replication, selectable marker, and the like. The regulatory sequences may be operably linked to each other to allow transcription and subsequent translation.

The modified peptide of the invention may also be modified to include such features as to ease the recovery of said polypeptide in production. One non- limiting example is the inclusion of Histidine tags, which are useful in Nickel column recovery. Peptides containing such a His-tag will be retained on a Nickel- chelator column. A second non-limiting example of a modification at gene level would be the inclusion of a biotin tag. Such biotin-labelled peptides would be retained on a streptavidin column.

Modified peptides of the invention may comprise D-amino acids. See also Definitions for examples.

Modified peptides of the invention may optionally be modified post- translationally. Post translational modification may include any of the following: Acteylation, alkylation, methylation, demethylation, amidation, biotinylation, formylation, carboxylation, glycosylation, lipoylation, iodination, hydroxylation, glycylation, isoprenylation, oxidation, phosphorylation, sulfation, deamidation and/ or deamination.

Accordingly the peptide of the present invention may be modified at the C-terminal part by amidation or esterification and at the N-terminal part by acylation, acetylation, PEGylation, acylation and the like, such as to increase stability within the mammal to be treated.

In addition to the modified peptides of the invention described above, sterically similar variants may be formulated to mimic the key portions of the peptide structure. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. Preferred fragments and modified peptides of the invention have retained at least some or all of the antimicrobial activity of the peptide of the invention, such as anti-bacterial, anti-fungal, anti-viral, anti-parasitic, anti-yeast or any combination of said activities.

Additionally the peptide may be operably linked to other known peptides or polypeptides or other substances, such other peptides, lipids, proteins, oligosaccharides, polysaccharides, other organic compounds, or inorganic substances. For example the peptides may be coupled to a substance which protects the peptides from being degraded prior to them having exerted their effect.

The invention also relates to a pharmaceutical composition which comprises at least one peptide of the invention as defined above and a

pharmaceutical acceptable adjuvant, carrier, buffer or diluent.

The invention also relates to a pharmaceutical composition which comprises the peptide as defined above and a pharmaceutical acceptable adjuvant, carrier, buffer or diluent.

The term "adjuvant" is intended to mean drugs that have few or no pharmacological effects by themselves, but can increase the efficacy or potency of other drugs when given at the same time. An adjuvant may also be an agent which, while not having any specific antigenic effect in itself, can stimulate the immune system, increasing the response to a medicament such as a vaccine. The adjuvant may for example be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. Adjuvants may furthermore be for example virosomes, squalene or others.

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tar- trate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The term "diluent" is intended to mean an aqueous or non- aqueous solution with the purpose of diluting the peptide in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

Proper formulation is dependent upon the route of administration chosen. The pharmaceutical compositions according to the invention may be administered locally or systemically. Routes of administration include topical, transdermal, ocular, nasal, pulmonar, buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral, parenteral, vaginal and rectal. Also administration from implants is possible.

One route of administration is topical. For topical administration, the peptides may be formulated as is known in the art for direct application to a target area, for example nails and skin. Forms chiefly conditioned for topical application take the form, for example, of lacquers, creams, milks, gels, powders, dispersion or micro-emulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g. sprays or foams), soaps, detergents, lotions or cakes of soap. Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols.

A second route of administration is by inhalation. Thus, the compositions for use according to the present invention may conveniently be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatine for use in an inhaler or insufflator may be formulated containing a power mix of the compound and a suitable powder base such as lactose or starch. Said embodiment is suitable for treatment of for example airway disease.

A third formulation is compositions formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

Further examples of suitable preparation forms are, for example granules, powders, tablets, coated tab-lets, (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 characterised by long-range order but short-range disorder (examples include lamellar, hexagonal and cubic phases, either water- or oil continuous), or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, aerosols, droplets or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients, diluents, adjuvants or carriers are customarily used as described elsewhere herein. .

Yet further examples of suitable preparation forms include suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatine, and polymers such as polyethylene glycols.

The pharmaceutical compositions of the invention may also be in the form of a liposome, in which the peptide is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglyc- erides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is can be found in for example US4,235,871.

The pharmaceutical compositions of the 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(carprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microshperes. Preparations of such micro- spheres can be found in US 5,851,451 and in EP0213303.

The pharmaceutical compositions of the invention may also be in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellu-lose, ethylhydroxyethyl cellulose, alginates,carageenans,hyaluronic acid and deriva-tives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene ox-ide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalco- hol/polyvinylacetate of different degrees of hydrolysis, and for example

polyvinylpyrrolidone amy be used for thickening of the solution containing the polypeptide.

Alternatively, the peptides may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers. The pharmaceutical composition may also include ions and a defined pH for potentiation of action of the antimicrobial polypeptides of the invention.

The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc..

The pharmaceutical composition may also comprises at least one other therapeutic agent or agents, such as antibiotic, anti-inflammatory, antiseptic agents, anti-fungal agents, anti-viral agents, anti-parasitic agents, or a combination thereof. Examples of anti-bacterial agents are are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide. Examples of anti-inflammatory agents are steroids and macrolactam derivatives. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying,

encapsulating, entrapping or lyophilizing processes.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a composition of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a disease.

The present invention relates in another aspect to the use of the polypeptide molecule according to the invention for the manufacturing of a medicament for the treatment or prevention of a microbial infection. Said

medicament may be directed to alleviating, preventing and/or curing infections caused by any single microorganism, or a combination of microorganisms.

Examples of bacteria (e.g., Gram-positive, Gram-negative), are

Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, as well as other Gram positive bacteria, Gram negative bacteria Enterococcus faecalis, Mycobacterium tuberculosis,

Mycobacterium bovis, Mycobacterium avium subsp. Paratuberculosis, Borrelia burgdorferi, other spirochetes, Helicobacter pylori, Streptococcus pneumoniae, Listeria monocytogenes, Proteus mirabilis, Finegoldia magna, Histoplasma capsulatum, Bartonella henselae, Bartonella quintana Citrobacter sp., Klebsiella sp., Enterobacter sp., Morganella, Providencia, Listeria sp., Salmonella sp., Serratia sp., Shigella sp., Yersinia sp., Pasteur ella sp., Vibrio sp., Campylobac-ter sp., k sp., Bordetella sp., Brucella sp., Neiserria sp., k sp., Mycoplasma sp., and Chalmydia sp. giving rise to clinical manifestations such as Tuberculosis,

Pneumonia, Stomach ulcers, Paratuberculosis and others. Examples of fungi are Aspergillus fumigatus, Candida albicans, Candida parapsilosi as well as Malassezia species. Cryptococcus neoformans, Pneumocystis carinii giving rise to clinical manifestations such as skin-, nail-, and mucosal infections, Meningitis, Sepsis and others; parasites (e.g., protozoans, nematodes, cestodes and trematodes); viruses, and prions. Specific organisms in these classes are well known (see for example, Davis et al, Microbiology, 3.sup.rd edition, Harper & Row, 1980).

The invention also relates to the use of the above defined peptides for the manufacturing of a medicament for the treatment of a bacterial, yeast or fungal disease or a mixture thereof. Said mixture of diseases relates to a disease or infection involving two or more species of one or more of bacteria, yeast or fungus. Examples of bacteria and fungi being shown above.

The invention thus relates also to the use of a peptide having a length of from 10 to 40 amino acid residues being derived from either SEQ IDNO: l or SEQ ID NO:4 or which has at least 75 % homology to SEQ ID NO: 1 or 4 or wherein SEQ ID NO: l or SEQ ID NO:4 has one or more substitutions for the manufacturing of a medicament for the treatment of a bacterial, yeast or fungal disease or a mixture thereof.

The invention also relates to said use, wherein said peptide is SEQ ID NO:2-3 or 5-31 or which has at least 75 % homology to SEQ ID NO:2-3 or 5-31 or wherein SEQ ID NO:2-3 or 5-31 has one or more substitutions. The invention further relates to said use, wherein said peptide has at least 80% homology to SEQ ID NO. 2-3 or 5-31. The invention further relates to said use. wherein said peptide is SEQ ID NO:2-3 or 5-31. The invention further relates to said use wherein said peptide is SEQ ID NO.2 (MK5), SEQ ID NO.15 (MK9), SEQ ID NO. 17 (MK1 1), SEQ ID NO. 3 (MK 12), SEQ ID NO.5 (PTN 5) or SEQ ID NO. 6 (PTN 12).

The invention may be useful in the treatment or prevention of disorders such as impetigo, erysipelas, bacterial and fungal wound infections, bacterial and fungal prosthesis infections, bacterial and fungal arthritis, bacterial and fungal conjunctivitis, bacterial and fungal pharyngitis, bacterial and fungal laryngitis, bacterial and fungal acute bronchitis, bacterial and fungal exacerbations in COPD and cystic fibrosis, bacterial and fungal pneumonia, bacterial and fungal gastritis, bacterial and fungal gastroenteritis, bacterial and fungal colitis, bacterial and fungal meningitis, bacterial and fungal cerebral abscess, bacterial and fungal external otitis, bacterial and fungal otitis media, bacterial and fungal osteomyelitis, bacterial and fungal pleural empyema, parodontitis, bacterial and fungal vulvitis, bacterial and fungal vaginitis, bacterial and fungal salpingitis, bacterial and fungal peritonitis, bacterial and fungal rhinitis, bacterial and fungal mucositis, bacterial and fungal urethritis, bacterial and fungal epididymitis, bacterial and fungal prostatitis, bacterial and fungal endocarditis, necrotizing fasciitis, and bacterial and fungal septicemia, infected acute wounds, chronic wounds, topical wounds, burn wounds including sunburn and thermal burns, ulcers such as chronic skin ulcers or stomach ulcers, paronychia, microbial keratitis, mastitis, acne,impetigo, infected skin eczema, atopic dermatitis, vaginal infections, external otitis, seborrhoic dermatitis, oral infections and parodontitis, candidal intertrigo, conjunctivitis and other eye infections such as P. aeruginosa keratitis; pneumonia and other airway diseases; and gastrointestinal disorders. In addition, diseases caused by viruses (e.g. HIV, Herpes simplex, Papilloma-viruses, and rhinoviruses).

The instant invention is also useful in the treatment and prevention of opportunistic or complicating secondary infections, for example in cystic fibrosis, after surgery and after skin trauma, in airway disease and in gastrointestinal disorders.

Furthermore, the peptides of the invention may also be used as sterilising or cleaning aids for use, for example, on surfaces to reduce and/or eliminate contamination by bacteria. For example, peptides of the present invention may be added to or diluted in an appropriate excipient or solution prior to use as a sterilising or cleaning agent. Such sterilising or cleaning solutions may be used to decontaminate, for example, furniture, floors, equipment including for example specialised hospital equipment and/or surgical equipment. In a further embodiment, the polypeptides of the invention may be used to eliminate and/or reduce bacterial or fungal contamination on parts of the body, particularly for example, the hands. The peptides of the invention may be diluted as an aqueous or non-aqueous solution (dissolved in aqueous, non aqueous or organic solvent) and which may be applied to a body part, for example the hands.

The pharmaceutical composition may also be included in solutions intended for storage and treatment of external materials in contact with the human body, such as contact lenses, orthopaedic implants, and catheters.

Additionally, the compositions may be used for prevention of infection post-surgery in plasters, adhesives, sutures, or be incorporated in wound dressings.

The peptides of the invention may also be used in polymers, textiles or the like to create antibacterial surfaces or cosmetics, and personal care products (soap, shampoos, tooth paste, anti-acne, suncreams, tampons, diapers, etc) may be supplemented with the pharmaceutical compositions.

Pharmaceutical compositions comprising the peptide of the invention and useful in treating or preventing disorders related to microbial infections are described above.

In a final embodiment the invention relates to a method of treatment comprising administering a pharmaceutical composition as defined above to a mammal suffering from a bacterial of fungal disease or a mixture thereof. Thus the methods are applicable to both human therapy and veterinary applications.

The mammal, suitable for such a treatment may be identified by well- established hallmarks of an infection, such as fever, pulse, culture of organisms, and the like.

Disorders and conditions that may be treated with the peptides of the invention include those caused by or aggravated by one or more types of

microorganisms.

The pharmaceutical compositions are preferably administered to a patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose is dependent on the, activity of the com-pound, manner of administration, nature and severity of the disorder, age and body weight of the patient different doses may be needed. Determination of the effective amounts is well within the capability of those skilled in the art.

The administration of the dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of sub-divided doses at specific intervals.

To achieve the desired effect(s), the peptides, may be administered as single or divided dosages, for example, of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight or at least about 1 mg/kg to about 20 mg/kg of body weight, although other dosages may provide beneficial results.

Following examples are intended to illustrate, but not to limit, the invention in any manner, shape, or form, either explicitly or implicitly.

EXAMPLES

Materials and Methods

Chemicals, reagents and methods

Recombinant human PTN and MK were from PeproTech (London, United Kingdom). According to the manufacturer, both MK and PTN have a correct set of intramolecular disulfide bonds. 20-mer peptides corresponding to various regions of human MK, PTN, and corresponding orthologues were from Sigma (PEP screen®).

Bacterial Strains and Growth Conditions The Streptococcus pyogenes strain API (40/58) of Ml serotype was from the World Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. Staphylococcus aureus (strain 5120), Escherichia coli (strain 37.4), and Pseudomonas aeruginosa (strain 27-1) were obtained from the Division of Infection Medicine, Department of Clinical Sciences, Lund University, Sweden. All bacteria were routinely grown in Todd- Hewitt (TH; Difco/Becton & Dickinson, Franklin Lakes, NJ) liquid medium at 37°C and 5% CO₂. Viable Count Assay

All bacteria were cultivated to mid-logarithmic phase (optical density at 620 nm was 0.4) in TH medium, washed, and diluted (1 : 1,000) in incubation buffer (10 mM Tris-HCl, containing 5 mM glucose; pH 7.4). P. aeruginosa were grown for 18 hours at 37°C. Fifty μΐ of bacteria (10⁶ colony forming units

(CFU)/ml) were incubated in the absence or presence of peptides at various concentrations, (0.003- ΙμΜ) for one hour at 37°C. To quantify bactericidal activity, serial dilutions of the incubation mixtures were plated on TH agar plates, incubated overnight at 37°C, and the number of CFU determined.

In order to investigate the activity of MK and PTN in the presence of salt, the viable counts assay was also performed after addition of different concentrations of sodium chloride to the incubation buffer (5-150 mM). The assay was also performed in the presence of human plasma (pooled citrated plasma from at least 12 donors; Department of Transfusion Medicine, University Hospital, Lund, Sweden) and sodium chloride (150 mM) at plasma concentrations ranging from 1% to 30%.

Liposome leakage assay

The liposomes investigated were anionic (DOPE/DOPG 75/25 mol/mol). DOPG (l,2-Dioleoyl-sn-Glycero-3-Phosphoglycerol, monosodium salt) and DOPE (l,2-dioleoyl-sn-Glycero-3-phoshoetanol-amine) were from Avanti

Polar Lipids (Alabaster, AL) and of >99% purity. The lipid mixtures were dissolved in chloroform, after which the solvent was removed by evaporation under vacuum overnight. Subsequently, 10 mM Tris buffer, pH 7.4, with or without additional 150 mM NaCl, was added together with 0.1 M carboxyfluorescein (CF) (Sigma, St Louis, MO). After hydration, the lipid mixture was subjected to eight freeze-thaw cycles consisting of freezing in liquid nitrogen and heating to 60°C. Unilamellar liposomes of about 0 140 nm were generated by multiple extrusions through polycarbonate filters (pore size 100 nm) mounted in a LipoFast mini-extruder (Avestin, Ottawa, Ontario, Canada) at 22°C. Untrapped CF was removed by two subsequent gel filtrations (Sephadex G-50, GE Healthcare, Uppsala, Sweden) at 22°C, with Tris buffer (or Tris plus 150 mM NaCl) as eluent. CF release from the liposomes was determined by monitoring the emitted fluorescence at 520 nm from liposome dispersions (10 mM lipid in 10 mM Tris with 5 mM glucose, pH 7.5), with or without 150 mM additional NaCl). An absolute leakage scale was obtained by disrupting the liposomes at the end of each experiment by addition of 0.8 mM Triton X-100 (Sigma), causing 100% release and dequenching of CF. A SPEX- fluorolog 1650 0.22-m double spectrometer (SPEX Industries, Edison, NJ) was used for the liposome leakage assay. Measurements were performed in at least duplicate at 37°C.

Radial Diffusion Assay

Radial diffusion assay (RDA) was performed essentially as described earlier (35). E. coli was grown to mid-logarithmic phase (optical density at 620 nm was 0.4) in 10 mL of full strength (3%, w/v) trypticase soy broth (TSB) (BD

Biosciences, San Jose, CaL). The bacteria were washed once with 10 mM Tris-HCl (containing 5mM glucose; pH 7.4). 4xl0⁶ CFU were added to 5 mL of the underlay gel (0.03% TSB, 1% low electro endosmosis-type agarose (Low-EEO; Sigma), 0.02% Tween-20 (Sigma) in 10 mM Tris-HCl) and poured onto a 0 90 mm Petri dish. After agarose solidification, 0 4 mm wells were punched and 6 μΐ of buffer alone or peptide (ΙΟΟμΜ) were added to each well. Plates were incubated at 37°C for 3h to allow the peptides to diffuse. 5 mL of the overlay gel (6% TSB, 1% low- EEO agarose in 10 mM Tris-HCl) was used to cover the underlay gel.

Antimicrobial activity was seen as a clearing zone around each well after incubating 18-24h at 37°C. The assay was performed in triplicates with either the recombinant human MK and PTN holopeptides or, overlapping peptides, (comprised of 20 amino acids) derived from the mature polypeptide sequences of MK and PTN (accession numbers NP_001012333 and CAA37121).

Viable counts assay

Bacteria were cultivated to mid-log phase (OD₆₂o 0.4) in TH, washed, and diluted in incubation buffer (10 mM Tris-HCl, containing 5 mM glucose; pH 7.4). Fifty μΐ of bacteria (10⁶ colony forming units (cfu)/mL) were incubated together with various concentrations of peptide or buffer alone for one hour at 37°C. To quantitate bactericidal activity, serial dilutions of the incubation mixtures were plated on TH agar. Plates containing less than 30 or more than 200 cfu were excluded from the experiment. Midkine Sequence (holoprotein):

VAKKKDKVKKGGPGSECAEWAWGPCTPSSKDCGVGFREGTCGAQTQRIR CRVPCNWKKEFGADCKYKFENWGACDGGTGTKVRQGTLKKARYNAQCQ ETIRVTKPCTPKTKAKAKAKKGKGKD

MK5: CGAQTQRIRCRVPCNWKKEF

MK 12 :KPCTPKTKAKAKAKKGKGKD

Pleiotrophin Sequence (holoprotein):

GKKEKPEKKVKKSDCGEWQWSVCVPTSGDCGLGTREGTRTGAECKQTMK TQRCKIPCNWKKQFGAECKYQFQAWGECDLNTALKTRTGSLKRALHNAE CQKTVTISKPCGKLTKPKPQAESKKKKKEGKKQEKMLD

PTN5: CKQTMKTQRCKIPCNWKKQF

PTN 12 :KPCGKLTKPKPQAESKKKKKEGKKQE

Bactericidal activity of synthetic fragments (20 amino acids)

MK1 : VAKKKDKVKK GGPGSECAEW: ++

MK2: GGPGSECAEW AWGPCTPSSK: +

MK3: AWGPCTPSSK DCGVGFREGT: +

MK4: DCGVGFREGT CGAQTQRIRC: +

MK5: CGAQTQRIRC RVPCNWKKEF: +++

MK6: RVPCNWKKEF GADCKYKFEN: +(+)

MK7: GADCKYKFEN WGACDGGTGT: +(+)

MK8: WGACDGGTGT KVRQGTLKKA: ++

MK9: KVRQGTLKKA RYNAQCQETI: ++(+)

MK10: RYNAQCQETI RVTKPCTPKT: ++

MK1 1 : RVTKPCTPKT KAKAKAKKGK: +++

MK12: KPCTPKT KAKAKAKKGK GKD: +++

Homo Sapiens - PTN:

PTN1 : GKKEKPEKKV KKSDCGEWQW: ++

PTN2: KKSDCGEWQW SVCVPTSGDC: +(+) PTN3: SVCVPTSGDC GLGT EGT T: +(+)

PTN4: GLGTREGTRT GAECKQTMKT: +

PTN5: GAECKQTMKT QRCKIPCNWK: ++(+)

PTN6: QRCKIPCNWK KQFGAECKYQ: ++

PTN7: KQFGAECKYQ FQAWGECDLN: +

PTN8: FQAWGECDLN TALKTRTGSL: +

PTN9: TALKTRTGSL KRALHNAECQ: +(+)

PTN10: KRALHNAECQ KTVTISKPCG: +(+)

PTN1 1 : KTVTISKPCG KLTKPKPQAE: ++

PTN 12 : KLTKPKPQAE SKKKKKEGKK: ++(+)

PTN13:PQAE SKKKKKEGKK QEKMLD: +

MBC₉₀:

0,1-1 μΜ: +++

1-10 μΜ: ++

10-30 μΜ: +

EXAMPLE 1

Midkine and pleiotrophin possess antibacterial activity

Recombinant human midkine and pleiotrophin were investigated for possible antibacterial activity using a viable count assay. Both peptides were highly antibacterial against the gram-positive bacteria Streptococcus pyogenes,

Staphylococcus aureus, and Streptococcus pneumoniae (Table I) and the gram- negative bacteria Escherichia coli and Pseudomonas aeruginosa (Table II) TABLE I

S. pyogenes S. aureus S. pneumoniae

MBC50 MBC₉₀ MBC50 MBC₉₀ MBC50 MBC₉₀ (μΜ) (μΜ) (μΜ) (μΜ) (μΜ) (μΜ)

Midkine 0,03 0,08 0,03 0,08 0,06 0,2

Pleiotrophin 0,06 ο,ι 0,16 0,28 0,07 0,15 The values represent mean from three independent experiments. MBC₅o and MBC₉₀ respectively are the peptide concentrations at which 50% and 90% of the bacteria were killed as detected by viable counts.

Table II

EXAMPLE 2

The antibacterial activity of midkine and pleiotrophin is resistant to sodium chloride at physiologic concentrations

Since the viability of S. aureus is not affected by the presence of NaCl at physiologic concentrations (i.e. 150 mM), the activity of MK and PTN were tested against this pathogen (Table III). A slight decrease of antibacterial activity with increasing concentrations of NaCl was observed. Table III

EXAMPLE 3

Midkine and pleiotrophin possess fungicidal activity

Both MK and PTN exert fungicidal activity against Candida albicans as demonstrated in Table IV. Table IV

(MFC: Minimal Fungicidal Concentration)

EXAMPLE 4

Human MK and PTN possess antibacterial activity.

MK and PTN were investigated for possible antibacterial activity against a panel of pathogenic bacteria, i.e. the gram-positive bacteria Streptococcus pyogenes and Staphylococcus aureus as well as the gram-negative bacteria, Pseudomonas aeruginosa, and Escherichia coli. The bacteria were incubated in buffer alone or with recombinant MK or PTN at concentrations ranging from 0.003 μΜ to 1 μΜ followed by a viable count assay. Both MK and PTN showed high bactericidal activity against Gram-positive bacteria (Figs. 1A & B) and slightly lower activity against Gram-negative bacteria (Figs. 1C & D).

Human MK and PTN possess fungicidal activity.

MK and PTN were investigated for fungicidal activity against Candida albicans and Candida parapsilosis . The fungi were incubated in buffer alone or with recombinant MK or PTN at concentrations ranging from 0.01 μΜ to 3 μΜ followed by a viable count assay. Both MK and PTN showed high fungicidal activity against both fungi (Figs. 4 A & B) Antibacterial activity of MK and PTN in presence of salt and plasma.

Physiological concentrations of sodium chloride impair the antimicrobial activity of many AMPs, such as defensins. Saliva, during fasting conditions, contains 5 mM sodium chloride, plasma has a concentration slightly below 150 mM, while epithelial surfaces have concentrations in between.

Therefore, we chose to study the bactericidal activity of MK and PTN in the presence of sodium chloride at concentrations ranging from 5-150 mM. Since S. pyogenes itself is sensitive to salt, S. aureus and E. coli were investigated. The antibacterial activity was largely unaffected, except in the case of S. aureus incubated with PTN, where a dose-dependent decrease of the bactericidal activity by 23% was seen at 150 mM sodium chloride (Fig. 2B). The presence of plasma is also known to inhibit the activity of AMPs. The influence of human citrated plasma on the killing capacity of MK and PTN was therefore investigated using S. aureus and E. coli in the viable count assay. Already at low concentrations of plasma, a strong inhibition of both MK- and the PTN-induced killing was seen, although less pronounced in the case of E. coli (Figs. 2C & D).

Fungicidal activity of MK and PTN in presence of salt.

Furthermore, the fungicidal activity of MK and PTN in the presence of salt was evaluated using viable count assay with addition of sodium chloride. While the fungicidal activity of MK against C. albicans was influenced by salt

concentration (Fig 5A), in the other cases fungicidal activity remained largely unchanged. (Fig 5 B- D).

MK and PTN have membrane-disrupting properties.

Many AMPs have a mode of action that includes disruption of the bacterial plasma membrane. To explore if this is the case for MK and PTN, model lipid bilayers were investigated in liposome leakage experiments. Addition of human recombinant MK and PTN caused leakage of fluorescent dye from micelles even at low concentrations of the polypeptides. Addition of sodium chloride at 150 mM did not affect the interaction between MK, PTN and the lipid bilayers (Figs. 3 A & B). Electron microscopy was used to investigate effects on the bacterial morphology and integrity during the killing process. S. pyogenes and E. coli were incubated in either buffer alone, with MK, or PTN (Fig. 3C). In analogy to the liposome results, bacteria incubated with MK and PTN, showed membrane protrusions and leakage of intracellular content, indicating damage to the bacterial membranes.

Antibacterial domains of human MK and PTN.

The MK and PTN holopeptides are antibacterial but some regions within the molecules may be of particular importance (Fig. 1). To map the regions of the molecules that contain the antibacterial activity, overlapping peptides, 20 amino acids in length, were synthesized. These were used to screen for antibacterial activity by radial diffusion assay for E. coli. The distribution of the antibacterial activity within the molecules is shown in supplemental Fig. 1. The regions with the highest antibacterial activity were localized in the last β-strand in the NH₂-terminal domain (peptides 5 and 6, i.e. MK5 and PTN5, and MK6 and PTN6 respectively), in the middle β-strand of the C-terminal domain (peptide 9, i.e MK9 and PTN9) and also in two peptides derived from the COOH-terminal tail (peptides 1 1 and 12, i.e. MK 1 1 and PTN 1 1, and MK 12 and PTN 12 respectively).

Fungicidal domains of human MK and PTN.

The fungicidal activity of peptides from MK was shown in radial diffusion assays (Fig 6A). MK5, MK9, MKl 1 and MK 12 were shown to have fungicidal activity. The fungicidal activity of these peptides was also demonstrated using viable count assays (Fig 7 A). The effect of salt on the peptides was demonstrated in viable count assays including sodium chloride in varying concentrations (Fig 7 B-E).

Claims

1. A peptide having a length of from 10 to 40 amino acid residues being derived from either SEQ ID NO: 1 or SEQ ID NO:4 or which has at least 75 % homology to SEQ ID NO: 1 or 4 or wherein SEQ ID NO: 1 or SEQ ID NO:4 has one or more substitutions.

2. The peptide according to claim 1, wherein said peptide is SEQ ID NO:2-3 or 5- 31 or which has at least 75 % homology to SEQ ID NO:2-3 or 5-31 or wherein SEQ ID NO:2-3 or 5-31 has one or more substitutions.

3. The peptide according to claims 1-2, wherein said peptide has 1, 2, 3 or 4 substitutions.

4. The peptide according to claim 2, wherein said peptide is SEQ ID NO. 2-3 or 5- 31.

5. The peptide according to claim 4; wherein said peptide is SEQ ID NO.2 (MK5), SEQ ID NO.15 (MK9), SEQ ID NO. 17 (MK1 1), SEQ ID NO. 3 (MK 12), SEQ ID NO.5 (PTN 5) or SEQ ID NO. 6 (PTN 12).

6. A pharmaceutical composition which comprises at least one peptide according to any of claims 1-5 and a pharmaceutical acceptable adjuvant, carrier, buffer or diluent.

7. A pharmaceutical composition which comprises the peptide according to any of claims 1-5 and a pharmaceutical acceptable adjuvant, carrier, buffer or diluent.

8. The pharmaceutical composition according to claims 6 to 7, wherein the composition also comprises at least one other therapeutic agent.

9. Use of a peptide having a length of from 10 to 40 amino acid residues being derived from either SEQ IDNO: l or SEQ ID NO:4 or which has at least 75 % homology to SEQ ID NO: 1 or 4 or wherein SEQ ID NO: 1 or SEQ ID NO:4 has one or more substitutions for the manufacturing of a medicament for the treatment of a bacterial, yeast or fungal disease or a mixture thereof.

10. Use according to claim 9, wherein said peptide is SEQ ID NO:2-3 or 5-31 or which has at least 75 % homology to SEQ ID NO:2-3 or 5-31 or wherein SEQ ID NO:2-3 or 5-31 has one or more substitutions.

1 1. Use according to claim 9 to 10, wherein said peptide has at least 80% homology to SEQ ID NO. 2-3 or 5-31

12. Use according to claim 9 to 1 1, wherein said peptide is SEQ ID NO:2-3 or 5-31.

13. Use according to claim 9 to 12, wherein said peptide is SEQ ID NO.2 (MK5), SEQ ID NO.15 (MK9), SEQ ID NO. 17 (MKl 1), SEQ ID NO. 3 (MK 12), SEQ ID NO.5 (PTN 5) or SEQ ID NO. 6 (PTN 12).

14. Use according to any of claims 9 to 13 wherein said disease is caused by one or more organisms selected from the group of Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida parapsilosis .

15. A method of treatment comprising administering a peptide according to any of claims 1 to 5 or a pharmaceutical composition of any of claims 6 to 8 to a patient suffering from a bacterial, yeast or fungal disease or a mixture thereof.