WO2004089986A1 - Peptide antimicrobien de la famille des transferrines - Google Patents

Peptide antimicrobien de la famille des transferrines Download PDF

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Publication number
WO2004089986A1
WO2004089986A1 PCT/NL2004/000250 NL2004000250W WO2004089986A1 WO 2004089986 A1 WO2004089986 A1 WO 2004089986A1 NL 2004000250 W NL2004000250 W NL 2004000250W WO 2004089986 A1 WO2004089986 A1 WO 2004089986A1
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Prior art keywords
peptide
antimicrobial
seq
leu
lys
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PCT/NL2004/000250
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English (en)
Inventor
Arie Van Nieuw Amerongen
Engelmundus Cornelis Ignatius Veerman
Jasper Groenink
Marieke Ilonka Arwen Van Der Kraan
Johannes Gerhardus Maria Bolscher
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Stichting Voor De Technische Wetenschappen
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Publication of WO2004089986A1 publication Critical patent/WO2004089986A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to antimicrobial peptides. More in particular it relates to antimicrobial peptides which may be derived from bovine lactoferrin and other members of the transferrin superfamily.
  • antimicrobial peptides from a variety of species have been identified and described.
  • cecropins which are found in insects (Bowman et ah, Ami Rev Microbiol (1987) 41:103) and the defensins, which are found in phagocytic cells from several mammalian species, including polymorphonuclear leukocytes from man (Ganz et al., Infect Immun (1987) 55:568). Some of them are derived from larger proteins, presumably by proteolytic degradation.
  • a well-known source of antimicrobial peptides is lactoferrin (LF), a multifunctional 80 kDa glycoprotein of mammalian origin.
  • WO 01/34641 discloses a polypeptide which includes 6 to 27 contiguous amino acids from the N-terminal segment of human lactoferrin.
  • US 6,399,570 discloses a 6 kDa host-defence polypeptide which is generated by proteolytic digestion of the lactoferrin molecule.
  • the 6 kDa host-defense polypeptide has antimicrobial activity and endotoxin-neutralizing activity.
  • Lactoferricin is produced in the stomach, when lactoferrin in milk is cleaved by pepsin. It has more potent bactericidal and fungicidal activity than the native lactoferrin protein.
  • Lactoferricin B obtained by pepsin digestion of bovine lactoferrin (bLF), consists of a positively charged looped peptide containing residues 17-41 of the native molecule. In addition to the corresponding loop, the human lactoferricin contains residues 1-17 as linear stretch, attached to the loop by a cysteine bridge. Lactoferricin B lacking the corresponding linear stretch is more potent than its human counterpart, however a peptide containing residues 1-17 of human lactoferricin is much more potent than its bovine counterpart and bovine lactoferricin.
  • lactoferricin B has been considered the sole antimicrobial domain of lactoferrin.
  • the Figure shows the antimicrobial activity towards C. alhicans of three peptides of the invention which all comprise a sequence according to SEQ ID NO.l
  • the peptide induced cellular uptake of propidium iodide (PI) was taken as a measure of antimicrobial activity.
  • Peptides which lacked the amino acid sequence of SEQ ID NO.l were taken as a control.
  • AU arbitrary units.
  • the present invention relates to a 17 to 50-mer antimicrobial peptide which comprises an amino acid sequence as depicted in SEQ ID No. 1 or an amino acid sequence which has at least 50% identity to the amino acid sequence of SEQ ID No.l and a net positive charge of at least +2.
  • Suitable examples of the peptide of the invention include, but are not limited to: (a) WKLLSKAQEKFGKNKSR (SEQ ID No.1)
  • antimicrobial peptides of the invention provide an alternative to existing antimicrobial peptides and antibiotics for treating and preventing microbial infections.
  • Another advantage of antimicrobial peptides of the invention is that the peptides may be prepared synthetically, but also enzymatically from members of the transferrin superfamily. A list of members of the transferrin superfamily is published, inter alia, in Crichton et.al. Eur J. Biochem. (1987) 164:485.
  • the amino acid sequence of SEQ ID No. 1 is present in native bovine lactoferrin (SEQ ID No. 6) where it corresponds to amino acids 268-284, but an identical or substantially similar sequence, i.e.
  • the peptides of the invention may show allelic variation and may be glycosylated and the glycosylation pattern may differ, if these peptides are prepared from a member of the transferrin superfamily.
  • allelic variants and glycosylated peptides are also part of the invention, irrespective whether they are derived from a member of the transferrin family or in another way.
  • bovine lactoferrin or “bovine lactoferrin” refers to a full length bovine lactoferrin polypeptide e.g. a polypeptide substantially as described by Moore et al. J Mol Biol 1997) 274:222).
  • An exemplary sequence for native bovine lactoferrin is the sequence of SEQ ID. No.6.
  • Yet another advantage of the peptides of the invention is that their action may be neutralised by disrupting the amino acid sequence of SEQ ID No. 1, which sequence is also present in SEQ ID No. 2, 3, 4 and 5.
  • One way in which this may be achieved is by enzymatic cleavage by pepsin, which enzyme will cleave bonds involving the aromatic amino acids phenylala ine, tryptophan, and tyrosine.
  • Another way is by using protease S. aureus V8 (endoproteinase Glu-C) which specifically cleaves peptide bonds on the carboxyterminal side of either aspartic or glutamic acids.
  • an "antimicrobial" peptide or compound may be any peptide or compound which possesses antimicrobial activity such as anti-bacterial, anti-fungal, anti-insecticidal, anti-parasitic and/or anti-viral activity.
  • a peptide is considered to have antimicrobial activity if its activity in a viability assay as described in section E of Materials & Methods is at least 10%, preferably at least 40, 50, 60 or 70%, most preferably at least 80% of the activity of any one of the peptides with a sequence which is identical to SEQ ID No. 1, 2, 3, 4, or 5.
  • the skilled person will understand that in general more peptide will have to be used if the peptide has less activity to obtain substantially the same effect.
  • Peptides of the invention encompass peptide variants.
  • Peptide variants comprise an amino acid sequence which is not identical but shows at least 50% identity to any of SEQ ID No. 1, 2, 3, 4 or 5, which has a net positive charge of at least +2 and which possesses antimicrobial activity.
  • the amino acid sequence shows at least 55%, 60%, 65%o, 70%), or 75%o identity.
  • the amino acid sequence shows at least 80%, 85%, 90% or 95% identity.
  • Percent sequence identity refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned.
  • 80% amino acid sequence identity means that 80% of the amino acids in two optimally aligned polypeptide sequences are identical.
  • a net positive charge of at least +2 is important since the introduction or presence of negatively charged amino acids may reduce antimicrobial activity. In particular replacement or deletion of each of the positively charged lysines (K) or arginines (R) will drastically reduce the antimicrobial activity.
  • peptides of the invention have a positive charge of at least +3.
  • peptides of the invention have a net positive charge of at least +4.
  • Peptides of the invention are not likely to have a net positive charge of more than +9.
  • polypeptides or variant polypeptides of the invention contain at least leucine (L 266) and isoleucine (1267).
  • the length of the peptide according to the invention should not exceed 50 amino acids.
  • a suitable size for a peptide according to the invention will be more than 12 and not more than 45 amino acids, preferably more than 15 but not more than 40 amino acids. Most preferred is a length of 17, 20, 26, 32 or 38 amino acids.
  • the total length of the peptide according to the invention should not exceed 50 amino acids when used as such, the peptide may be incorporated in another peptide e.g. for screening or (recombinant) production purposes, resulting in a longer peptide. This longer peptide sequence may comprise spacers and the like for allowing coupling to carriers or to other screening aids. Also derivatives of the peptides of the invention are enclosed within the scope of the invention.
  • Derivatives of the peptide of the invention are, for example, where functional groups, such as amino, hydroxyl, mercapto or carboxyl groups, are derivatized, e.g. glycosylated, acylated, amidated or esterified, respectively.
  • an oligosaccharide is usually linked to asparagine, serine and/or threonine.
  • Further derivatives are salts, especially pharmaceutically acceptable salts, such as alkali metal and alkaline earth metal salts, e.g. sodium, potassium, magnesium, calcium or zinc salts, or ammonium salts formed with ammonia or a suitable organic amine, such as a lower alkylamine, e.g.
  • the peptide may also be fused to fusion proteins. Fusion proteins and their production are well-known in the art and include, but are not limited to glutathion-S-transferase, maltose binding protein, metal binding polyhistidine, green fluorescent protein, luciferase, beta galactosidase.
  • the peptide of the invention may also be coupled to non- protein carrier, tags or labels that facilitate tracing of the peptide and allow for the identification and quantification of binding of the peptide to a substrate.
  • Peptides of the invention may be produced by isolation from any of the sources mentioned herefore and hereafter, especially from a larger protein or polypeptide (see Example 2), or from a cell expressing and/or secreting the peptide, it may be chemically synthesized (see Example 1), or may be expressed from a recombinant nucleic acid molecule. Following chemical synthesis or recombinant expression, the peptide may be produced as a precursor peptide and, therefore, may be further subjected to modifications, such as e.g. glycosylation and sialylation to yield the final peptide.
  • peptide synthesis which basically involves the steps of synthesizing a linear peptide of an amino acid sequence corresponding to the amino acid sequence of an antimicrobial peptide of the invention (see e.g. SEQ ID No. 1, 2, 3, 4 or 5) are well- known in the art.
  • peptides of the invention may be synthesized by hand or automatically using various techniques. For example, by using solid-phase techniques, such as Fmoc, which uses 9-fluorenylmethyloxycarbonyl for N- ⁇ amino protection and t-Boc, which uses tertiary butoxylcarbonyl.
  • solid-phase techniques such as Fmoc, which uses 9-fluorenylmethyloxycarbonyl for N- ⁇ amino protection
  • t-Boc which uses tertiary butoxylcarbonyl.
  • Roberge J. Y. et al.
  • Automated synthesis may be achieved, for example, using a Milligen/Biosearch 9050 peptide synthesiser (Millipore Corp., Bedford, MA, USA) or an Applied Biosystem 43XA series Peptide Synthesiser (Perkin Elmer).
  • peptides are synthesized by solid-phase peptide synthesis using Fmoc chemistry on a Milligen Biosearch 9050 peptide synthesiser (Millipore Corp., Bedford, MA, USA) according to the manufacturer's instructions.
  • a parent molecule which is a member of the transferrin superfamily (see Example 2), such as lactoferrin and transferrin, which may be found in a variety of organisms, in particular in vertebrates (Welch S.A. Comp Biochem Physiol (1990), 97B: 417).
  • the transferrin superfamily may be found in extracellular secretions such as sweat, tears, milk and saliva.
  • members of the transferrin superfamily may be found in oviducts secretions such as eggs.
  • an antimicrobial peptide of the invention was prepared by enzymatic digestion of bovine lactoferrin with an enzyme that cleaves at the C-terminal site of arginine such as endoproteinase Arg-C.
  • an antimicrobial peptide of the invention was prepared by enzymatic digestion of bovine lactoferrin with an enzyme that cleaves at the N-terminal site of asparagine, such as endoproteinase Asp-N.
  • An antimicrobial peptide of the invention may also be prepared by enzymatic digestion of bovine lactoferrin with a combination of enzymes. The enzymes may be working all at the same time or sequentially.
  • bovine lactoferrin is sequentially digested by endoproteinase Asp-N followed by endoproteinase Arg-C. The skilled person will understand that sequential digestion may be necessary, depending on the type and combination of enzymes used.
  • enzymes function only in the presence of EDTA, whereas others do not function in the presence of EDTA.
  • a combination of these two types of enzymes will dictate the order in which the enzymes are used.
  • the enzymes may be used according to the manufacturer's instructions. Usually, even less enzyme may be used.
  • the amount of enzyme used is between 1:1000 to 1:20 of the protein by weight, the incubation time between 1 and 18 hours at about 37°C depending on the amount of enzyme used. In a preferred embodiment, 1:500 to 1:1000 (w/w) enzyme is incubated for 5-8 hours at 37°C.
  • the protein from which the peptide of the invention is prepared may be pre-treated using methods in the art. For example denaturation, dissociation and solubilisation of the protein may be enhanced by including agents such as SDS, DTT, urea, guanidin-HCI or acetonitrile to the reaction mixture.
  • a nucleotide fragment comprising a nucleotide sequence encoding a peptide of the invention is also encompassed by the present invention.
  • Peptides of the invention may be prepared from a nucleic acid molecule by any convenient method.
  • a nucleic acid molecule encoding the peptide of the invention may be chemically synthesized using routine methods or, if desired, can be purchased from a commercial source.
  • cDNA may be prepared synthetically using a DNA synthesiser or be isolated from a cDNA source, optionally after first producing it from an RNA source using reverse transcriptase.
  • RNA source e.g., a total human or bovine RNA or from a cDNA library using methods known in the art to recover the nucleotide sequence.
  • Recombinant DNA sequences in accordance with the invention may be prepared entirely from using publicly and commercially available materials.
  • a nucleic acid molecule encoding a peptide of the invention will be isolated, and optionally, purified.
  • nucleic acid molecule when used in reference to a nucleic acid molecule, means that the nucleic acid molecule is relatively free of proteins, lipids, nucleic acids or other molecules with which it is normally associated in a cell.
  • an isolated nucleic acid molecule encoding a peptide of the invention constitutes at least about 75% by weight of a sample containing the nucleic acid molecule, and usually constitutes about 90%) of a sample, particularly about 95%> of the sample or more.
  • an isolated nucleic acid molecule encoding a peptide of the invention may be contained in a vector.
  • nucleic acid molecule in a vector is not considered to be part of a sample when determining the degree of isolation of the nucleic acid molecule encoding the peptide of the invention, since the encoding nucleic acid molecule generally can be readily purified from the vector.
  • the nucleic acid molecule is said to be “purified” when it is present in a particular composition in a higher concentration than in its original environment, or if it is in combination with components not normally present upon expression from a naturally occurring or wild type organism.
  • the nucleotide sequence encoding the peptide of the invention is typically expressed in a suitable host cell using a vector.
  • a vector comprising a nucleic acid molecule encoding a peptide of the invention is therefore included in the scope of the invention.
  • the vector may for example be a plasmid, cosmid, phage or virus.
  • Vectors will frequently include one or more selectable markers to enable selection of cells transformed with them and, preferably, to enable selection of cells harbouring vectors incorporating heterologous DNA. Appropriate start and stop signals will generally be present. Additionally, if the vector is intended for expression, sufficient regulatory sequences to drive expression will be present. Vectors not including regulatory sequences are useful as cloning vectors. Any host may be used, as long as the product does not kill the host.
  • Suitable hosts which facilitate manipulation include but are not limited to Escherichia coli, Aspergillus niger, Streptococci, Saccharomyces cerevisiae, plants from the Solanaceae (tobacco, potato), Chinese hamster ovary cells, COS cells (Cercopithecus aethiops, African green monkey).
  • Escherichia coli will usually be the prokaryotic organism of choice.
  • yeasts such as Saccharomyces may be suitable, the different glycosylation patterns of yeasts may mean that other eukaryotic expression hosts are preferred.
  • the host cell may be part of a multicellular organism such as a transgenic plant or animal, preferably a non-human animal. Methods for generating transgenic animals are well-known in the art. Insect cells, such as those transfected with baculovirus, may be used as may mammalian cells such as COS cells, which could for example be SV40-transfected. Another aspect of the invention is therefore a host cell transfected or transformed with a nucleic acid molecule of the invention. Typically, the host cell will be cultured in a suitable nutrient medium under conditions which promote the formation of an antimicrobial peptide of the invention. Depending on the culture system used, the peptide will be secreted in the culture medium or be retained in the host cell. The method for producing the antimicrobial peptides of the invention using a host cell is also within the scope of the claims.
  • the peptide of the invention may be isolated and, optionally, purified, but this is not strictly necessary for it to exerts its action.
  • the peptide of the invention is not even isolated from the hydrolysate in which it was prepared.
  • a hydrolysate comprising the peptide is part of the invention.
  • Suitable hydrolysates may be made from e.g. dairy products, especially from milk products, which include, whole milk, skimmed milk, semi-skimmed milk, pasteurised milk, whey, whey protein concentrate, dried milk powder and fermented milk products.
  • chromatography such as affinity chromatography and gel permeation chromatography, ion-exchange chromatography such as anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, high performance liquid chromatography (FiPLC) and lectin chromatography, ammonium sulfate; precipitation such as ethanol precipitation; filtration such as ultra filtration, microfiltration or cross-flow filtration and extraction or a combination of these techniques.
  • the product may be dried after isolation and/or purification such as e.g., by freeze-drying or spray drying.
  • isolated or “purified” when used in reference to the peptide of the invention, means that the peptide is relatively free of proteins, lipids, nucleic acids or other molecules to which it is normally associated in its original environment (e.g., the natural environment if it is naturally occurring).
  • the material is said to be “purified” when it is present in a particular composition in a higher concentration than in its original environment, or if it is in combination with components not normally present upon expression from a naturally occurring or wild type organism.
  • a naturally occurring peptide present in a living organism is not isolated, but the same peptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such peptides could be part of a composition, and still be isolated in that such composition is not part of its original environment.
  • an isolated peptide of the invention constitutes at least about 75% by weight of a sample containing the peptide, and usually constitutes about 90% of a sample, particularly about 95%> of the sample or 99%> or more.
  • the peptide of the invention may also be used for the production of antibodies.
  • antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies.
  • An antibody, or antigen binding fragment of such an antibody is characterized by having specific binding activity for the peptide of at least about lx 10 5 M "1 .
  • Fab, F(ab') 2 , Fd and Fv fragments of the antibody which retain specific binding activity for an antimicrobial peptide of the invention, are included within the definition of an antibody.
  • antibody includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof.
  • non-naturally occurring antibodies may be constructed using solid phase peptide synthesis, may be produced recombinantly or may be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science (1989) 246:1275.
  • These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well-known to those skilled in the art.
  • An antibody of the invention is useful, for example, for screening for antimicrobial peptides, i.e., for determining the presence or level of an antimicrobial peptide of the invention in a sample, or for cloning a nucleic acid molecule encoding an antimicrobial peptide of the invention from an appropriate expression library.
  • An antibody of the invention may also be used to substantially purify an antimicrobial peptide of the invention from a sample, for example, following expression of an antimicrobial peptide of the invention from a recombinant nucleic acid molecule.
  • an antibody raised against an antimicrobial peptide of the invention may be used to screen an expression library to identify a clone containing a cDNA encoding the antimicrobial peptide of the invention. Therefore, a method for screening for antimicrobial components in a sample of interest is also within the scope of the invention. This method typically comprises the detection of a component which binds to an antibody or to a nucleotide fragment of the invention, whereby binding is indicative of antimicrobial activity, Suitable samples of interest are samples which will or are likely to contain the peptide of the invention and have been mentioned above.
  • An antimicrobial peptide of the invention or an antibody of the invention may be labeled so as to be detectable using methods well-known in the art.
  • the peptide or antibody can be labeled with various detectable moieties including a radiolabel, an enzyme, biotin or a fluorochrome.
  • Reagents for labeling a peptide or antibody may be included in a kit containing the peptide or antibody or may be purchased separately from a commercial source.
  • the invention further provides a kit which contains an antimicrobial peptide of the invention or an antibody of the invention or both.
  • kit may also contain a reaction cocktail that provides the proper conditions for performing an assay, for example, an ELISA or other immunoassay for determining the level of expression of an antimicrobial peptide of the invention in a sample, and may contain control samples that contain known amounts of an antimicrobial peptide of the invention and, if desired, a second antibody specific for the antibody of the invention.
  • kit may include a simple method for detecting the presence or amount of antimicrobial peptide of the invention in a sample that is bound to the antibody.
  • polyclonal antibodies for example, in a rabbit, goat, mouse or other mammal
  • monoclonal antibodies may be obtained using methods that are well-known and routine in the art.
  • spleen cells from a mouse immunized, for example, with an antimicrobial peptide of the invention having the amino acid sequence of SEQ ID NO.l may be fused to an appropriate myeloma cell line to produce hybridoma cells.
  • Cloned hybridoma cell lines may be screened using labeled antimicrobial peptide of the invention to identify clones that secrete monoclonal antibodies of the invention.
  • Hybridomas expressing monoclonal antibodies of the invention having a desirable specificity and affinity may be isolated and utilized as a continuous source of the antibodies, which are useful, for example, for preparing standardized kits as described above.
  • a recombinant phage that expresses, for example, a single chain antibody also provides a monoclonal antibody that may be used for preparing standardized kits.
  • a method of the invention encompasses administering to the environment an effective amount of an antimicrobial peptide of the invention such that the antimicrobial peptide, optionally in combination with other antimicrobial peptides of the invention, may reduce or inhibit the ability of the microorganism to grow or survive.
  • One or more peptides of the invention may be combined with other antimicrobial compounds, such as for example with antibiotica or antimicotica (Helmerhorst et al. (1999), Antimicrob. Agents Chemother. 43:702). This may be very advantageous if a synergistic effect is desired.
  • An antimicrobial peptide of the invention may be used in a variety of procedures for reducing or inhibiting the survival or growth of microorganisms, including the microbicidal inhibition of survival of a microorganism as well as the microbistatic inhibition of growth.
  • An antimicrobial peptide of the invention may also be used to detach harmful microorganisms.
  • the term "effective amount" refers to the amount of antimicrobial peptide of the invention that reduces or inhibits the survival or growth of a microorganism in an environment.
  • an effective amount of an antimicrobial peptide of the invention produces only minimal effects against the environment, although the level of an acceptable deleterious effect is weighed against the benefit caused by the antimicrobial effect.
  • an antimicrobial peptide of the invention may be incorporated into a delivery system such as liposomes, if desired. Furthermore, an antimicrobial peptide of the invention may be administered orally to a subject. In addition, an antimicrobial peptide of the invention may be administered topically to an environment, which may be a human subject, or may be placed in a solution. Although an antimicrobial peptide of the invention is generally effective in microgram per ml amounts, an effective amount for administration to a particular environment will depend, in part, on the environment. For example, when administered to a mammal such as a human, an antimicrobial peptide of the invention, in addition to having antimicrobial activity, may have an undesirable side effect.
  • an effective amount of an antimicrobial peptide of the invention will vary depending, e.g., on the characteristics of the target microorganism, the extent of prior infection or growth and the specific antimicrobial peptide of the invention that is administered. Also, an effective amount depends on the form in which the antimicrobial peptide of the invention is administered. For example, encapsulation or incorporation of another antimicrobial peptide may allow administration of a higher amount of the peptide than "free" peptide without producing unacceptable side effects.
  • an antimicrobial peptide of the invention may be used, for example, as a food preservative, a disinfectant or wound care compositions or a medicament or therapeutic agent.
  • compositions comprising a peptide of the invention or a nucleotide fragment of the invention are also encompassed in the invention.
  • Food and food products may be treated with an antimicrobial peptide of the invention for the purpose of preserving the food, preventing microbial contamination or outgrowth, detaching harmful microorganisms or eliminating or preventing infection by microorganisms.
  • growth or survival of microorganisms on meat in slaughter houses may be reduced or inhibited by contacting the product with the antimicrobial peptide of the invention.
  • the antimicrobial peptide may also be applied in or on processed meat products like steaks, hamburgers, sausages, salami's fillets and poultry products, such as chicken and turkey; on fish products and milk products such as drinking milk, fermented milk and dessert products.
  • Food crops such as fruits, vegetables and grains may be treated with an antimicrobial peptide of the invention in order to reduce or inhibit post-harvest spoilage caused by microorganisms.
  • transgenic plants or animals useful in the food industry may be produced by introducing a nucleic acid molecule encoding a precursor of or an antimicrobial peptide of the invention itself into the germline cells of such organisms. Methods for producing transgenic plants and animals are well-known and routine in the art.
  • the antimicrobial peptide of the invention may also be used as an additive in food, particularly for prophylactic purposes, e.g. in functional foods.
  • the antimicrobial peptide of the invention is administered to non-human animals, this is typically done by addition to the feeding-stuffs, which besides the composition of the invention may contain commonly used nutrients.
  • An antimicrobial peptide of the invention may be used as a disinfectant to reduce or inhibit the survival or growth of microorganisms on an object or in a solution, such as in a wound care composition. In fact, it may be used to treat essentially any object or solution that can sustain microbial growth, where the survival or growth of the microorganisms is undesirable.
  • One example is the use of the peptide of the invention in a cosmetic product, but any object, surface or solution, e.g., working benches, medical instruments, in particular, surgical instruments, the eye, mouth, ear or skin may be treated with an antimicrobial peptide of the invention.
  • the antimicrobial peptide of the invention may be applied topically to the object or the surface or may be added to the solution.
  • the antimicrobial peptide of the invention may also be used in a dental care or oral care composition.
  • a dental care or oral care composition include, but are not limited to, a mouth wash, mouth rinse, toothpaste, mouth spray, topical oral gel, chewing gum, teeth whitener and a dental or denture cleanser.
  • a mouth wash, mouth rinse, toothpaste, mouth spray, topical oral gel, chewing gum, teeth whitener and a dental or denture cleanser In addition to the peptide of the invention it will also comprise an orally acceptable adjuvant and an orally acceptable carrier.
  • the peptide of the invention may be used for the preparation of a medicament for the treatment or prevention of a microbial infection in any part of the human or animal body, including but not limited to a microbial infection of the oral cavity, the respiratory system, the digestive system, the reproductive system, the skin, the bloodstream, the ear or the eye.
  • a pharmaceutical composition the peptide of the invention will generally be present in association with a carrier or excipient and, optionally, a pharmaceutically acceptable adjuvant.
  • a pharmaceutically acceptable carrier may contain physiologically acceptable compounds that act, for example, to stabilize or increase the absorption of the antimicrobial peptide of the invention.
  • physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans; antioxidants such as ascorbic acid or glutathione; chelating agents such as EDTA, which disrupts microbial membranes; divalent metal ions such as calcium or magnesium; low molecular weight proteins; or other stabilizers or excipients.
  • carbohydrates such as glucose, sucrose or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such as EDTA, which disrupts microbial membranes
  • divalent metal ions such as calcium or magnesium
  • low molecular weight proteins low molecular weight proteins
  • Administration may be topical pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral.
  • Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration.
  • Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present invention which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well-known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s).
  • the peptide of the invention is normally administered together with a carrier, which may be a solid, semi-solid or liquid diluent or a capsule.
  • a carrier which may be a solid, semi-solid or liquid diluent or a capsule.
  • the compound may be mixed with a solid, pulverulent carrier.
  • these carriers include lactose, saccharose, sorbitol, lactitol, mannitol, starch such as potato starch or corn starch, amylopectin, cellulose derivatives or gelatin.
  • Other components may include an antifriction agent such as magnesium stearate, calcium stearate, polyethylene glycol waxes or the like.
  • the composition may be pressed into tablets.
  • Multiple-unit-dosage granules may also be prepared. Tablets and granules of the above cores can be coated with concentrated solutions of sugar, etc.
  • the cores may also be coated with polymers which change the dissolution rate in the gastrointestinal. Such polymers are hydroxypropylmethyl cellulose phtalate, cellulose acetate phtalate, and are commercially available.
  • the pharmaceutical preparations of the invention may be controlled release preparations which are typically prepared using cross-linked starches or sodium starch glycolates as a releasing matrix.
  • Gelatin capsules may be prepared in the form of soft or hard capsules. In the former case the active compound is mixed with an oil, and in the latter case, multiple-unit- dosage granules may be contained therein.
  • Liquid preparations for oral administration can be present in the form of syrups or suspensions, e.g., solutions containing from about 0.2% by weight to about 20% by weight of the active compound disclosed, and glycerol and propylene glycol. If desired, such preparations can contain coloring agents, flavoring agents, saccharin, and carboxymethyl cellulose as thickening agent.
  • the daily dose of the active compound and the number of applications per 24 hours may vary and are dependent on the way of administration.
  • the pharmaceutical composition of the invention may comprise one or more peptides of the invention or one or more nucleic acid molecules encoding one or more peptides of the invention optionally in combination with other antimicrobial products.
  • Escherichia coli K12 and Escherichia coli 0157:H7, Bacillus subtilis ATCC 9372 and Pseudomonas aeruginosa were cultured aerobically in Brain Heart Infusion (BHI), Tryptic Soy Broth (TSB) or Luria-Bertani medium (LB-medium; Difco) at 37°C.
  • Bacteria were also cultured on blood agar plates containing 5 mg/l Haemin and 1 mg/l Menadion or on LB-agar plates.
  • Candida albicans 315 was cultured aerobically at 30°C in Sabouraud dextrose broth (SDB; Difco) and on Sabouraud dextrose agar plates (SDA).
  • Peptides were synthesized by solid-phase peptide synthesis using Fmoc-chemistry on Miligen/Biosearch 9050 peptide synthesizer (Millipore Corporation, Bedford, MA, USA) according to the manufacturer's instructions. Subsequently, the peptides were purified to more than 95% purity using HPLC with a variable wavelength UV-detector (Jasco, Tokyo, Japan) with a semi-preparative reversed phase column (20 cm x 1 cm ID, 12 micrometer particles) using a discontinuous gradient.
  • a variable wavelength UV-detector Jasco, Tokyo, Japan
  • a semi-preparative reversed phase column (20 cm x 1 cm ID, 12 micrometer particles
  • endoproteinase Asp-N (sequencing grade) from Pseudomonas fi'agi
  • endoproteinase Arg-C sequencing grade
  • Antimicrobial activity was determined by monitoring the peptide-induced cellular uptake of propidium iodide (PI) (Molecular Probes Inc., Eugene, OR, USA).
  • PI propidium iodide
  • PPB potassium phosphate buffer, pH 7.0
  • albicans suspension or bacteria suspension (approximately 1.8*10 7 cells/ml) in 1 mM PPB was added to each well to yield a final volume of 200 ⁇ l per well.
  • Fluorescence indicating PI uptake and binding to DNA, was measured at different time points using a fluorescence reader (Fluostar Galaxy, BMG Labtechnologies, Offenburg, Germany).
  • Control experiments involved incubation of cells with PI in the absence of peptides, or in the presence of inactive control peptides, e.g. cystatin Sl-14.
  • the number of colony forming units (CFUs) in each well was determined as described below.
  • peptides were diluted 2-fold serially in 1 mM PPB, pH 7.0, starting with a peptide concentration of 100 ⁇ M.
  • Bacteria or yeast cells cultured as described above, were washed 3 times and set to a suspension of approximately 6.4* 10 6 cells/ml for C. albicans and OD ⁇ oo of 0.5-1.0 for the bacteria. Of this suspension 50 ⁇ l was added to each well to yield a final volume of 100 ⁇ l per well.
  • Peptide WKLLSKAQEKFGKNKSR (SEQ ID No. 1) corresponding to amino acids 268 - 284 of SEQ ID No. 6;
  • Peptide DLIWKLLSKAQEKFGKNKSR (SEQ ID No. 2) corresponding to amino acids 265 - 284 of SEQ ID No. 6 and which would be produced upon digestion of SEQ ID No. 6 with a combination of endoproteinase Asp-N and endoproteinase Arg-C;
  • Peptide DLIWKLLSKAQEKFGKNKSRSFQLFGSPPGQR (SEQ ID No. 4) corresponding to amino acids 265 - 296 of SEQ ID No. 6 and which would be produced upon digestion of SEQ ID No. 6 with endoproteinase Asp-N; and the following control peptide mixtures:
  • Peptides of the invention and control peptides were tested for their antimicrobial activity towards C. albicans.
  • the peptide induced cellular uptake of propidium iodide (PI) was taken as a measure of antimicrobial activity.
  • the results (Fig. 1) show that the synthetically produced peptides of the invention possess antimicrobial activity.
  • Their antimicrobial activity towards C. albicans is several thousands times higher than the activity of the control peptides mixtures which showed no antimicrobial activity against C. albicans.
  • Example 2 Antimicrobial activity of an Asp-N hydrolysate
  • Native bovine lactoferrin was pre-treated by heating 10 mg/ml bovine lactoferrin in 50 mM sodium phosphate buffer at pH 7.9 in the presence of 0.02% SDS (w/v) for 5 minutes at approximately 70°C. It was made sure that the temperature did not exceed 80°C. Then, the pre-treated protein was incubated for approximately 6 hours at 37°C at pH 7.9 with endoproteinase Asp-N in an amount of 1:1000 of the protein by weight. This hydrolysate was tested for its antimicrobial activity towards C. albicans.
  • PI propidium iodide
  • Table 1 LC 50 values" of LFampin 268-284 and LFampin 265-284.
  • LC 50 The concentration of peptide ( ⁇ M) resulting in 50% reduction of the 'viable counts' ' highest concentration tested.
  • Example 4 Antimicrobial activity of peptide variants
  • the antimicrobial activities of synthetic substitution and deletion variant peptides based on SEQ ID NO.2 were evaluated against C. albicans. Results are shown in Table 2.
  • the results of the deletion variants show that deletion of amino acids 279 - 284 drastically increases L 50 values (lower candidacidal activity).
  • the results of the substitution variants show that subsititution of the positivley charges amino acids lysine (K) or arginine (R) by glycine drastically reduces antimicrobial activity. Similar results are obtained if the neutral leucine (L 266) or isoleucine (I 267) are replaced.

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Abstract

La présente invention porte sur un nouveau peptide antimicrobien pouvant être obtenu à partir de la lactoferrine bovine ou d'un autre membre de la superfamille des transferrines. L'invention porte sur des molécules d'acide nucléique isolées codant le peptide antimicrobien, ainsi que sur des vecteurs, des cellules hôtes et des méthodes de recombinaison visant à produire le peptide antimicrobien. L'invention porte sur des hydrolysats comprenant le peptide antimicrobien de l'invention et sur des anticorps et sur des méthodes de diagnostic permettant de détecter plusieurs peptides antimicrobiens de l'invention.
PCT/NL2004/000250 2003-04-14 2004-04-14 Peptide antimicrobien de la famille des transferrines WO2004089986A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006131586A1 (fr) 2005-06-08 2006-12-14 Consejo Superior De Investigaciones Científicas Peptides bioactifs identifies dans des hydrolysats enzymatiques de caseines lactees et procede d'obtention
WO2017025846A1 (fr) * 2015-08-11 2017-02-16 Farmagens Health Care Srl Fragment de lactoferrine destiné à être utilisé en tant qu'agent antibactérien et antiviral
EP2650304B1 (fr) * 2010-12-06 2017-04-19 Korea Advanced Institute Of Science And Technology Copolymère multi-bloc peptidique antimicrobien s'exprimant sur la surface des cellules
CN112646026A (zh) * 2020-12-24 2021-04-13 四川大学 基于乳铁蛋白的仿生抗菌功能多肽及制备方法和应用

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WO1994004171A1 (fr) * 1992-08-11 1994-03-03 President And Fellows Of Harvard College Peptides immunomodulateurs
WO2001072322A2 (fr) * 2000-03-27 2001-10-04 Pharming Intellectual Property B.V. Administration parenterale de fortes doses de lactoferrine

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WO1994004171A1 (fr) * 1992-08-11 1994-03-03 President And Fellows Of Harvard College Peptides immunomodulateurs
WO2001072322A2 (fr) * 2000-03-27 2001-10-04 Pharming Intellectual Property B.V. Administration parenterale de fortes doses de lactoferrine

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DATABASE EMBL [online] 2 August 2000 (2000-08-02), SONSTEGARD ET AL.: "161767 BARC 5BOV Bos taurus cDNA 5', mRNA sequence.", XP002254594, Database accession no. BE477827 *
DIONYSIUS D A ET AL: "Antibacterial peptides of bovine lactoferrin: Purification and characterization.", JOURNAL OF DAIRY SCIENCE, vol. 80, no. 4, 1997, pages 667 - 674, XP002254593, ISSN: 0022-0302 *
GROENINK J ET AL: "CATIONIC AMPHIPATHIC PEPTIDES, DERIVED FROM BOVINE AND HUMAN LACTOFERRINS, WITH ANTIMICROBIAL ACTIVITY AGAINST ORAL PATHOGENS", FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, vol. 179, no. 2, 15 October 1999 (1999-10-15), pages 217 - 222, XP000997948, ISSN: 0378-1097 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006131586A1 (fr) 2005-06-08 2006-12-14 Consejo Superior De Investigaciones Científicas Peptides bioactifs identifies dans des hydrolysats enzymatiques de caseines lactees et procede d'obtention
EP2495250A2 (fr) 2005-06-08 2012-09-05 Consejo Superior de Investigaciones Cientificas Peptides bioactifs identifiés dans des hydrolysats enzymatiques de caséine du lait et procédé d'obtention
EP2650304B1 (fr) * 2010-12-06 2017-04-19 Korea Advanced Institute Of Science And Technology Copolymère multi-bloc peptidique antimicrobien s'exprimant sur la surface des cellules
US10406204B2 (en) 2010-12-06 2019-09-10 Korea Advanced Institute Of Science And Technology Multimeric antimicrobial peptide complex which is displayed on cell surface
WO2017025846A1 (fr) * 2015-08-11 2017-02-16 Farmagens Health Care Srl Fragment de lactoferrine destiné à être utilisé en tant qu'agent antibactérien et antiviral
CN112646026A (zh) * 2020-12-24 2021-04-13 四川大学 基于乳铁蛋白的仿生抗菌功能多肽及制备方法和应用

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