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WO2008012066A2 - Nouveau peptide - Google Patents

Nouveau peptide Download PDF

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Publication number
WO2008012066A2
WO2008012066A2 PCT/EP2007/006567 EP2007006567W WO2008012066A2 WO 2008012066 A2 WO2008012066 A2 WO 2008012066A2 EP 2007006567 W EP2007006567 W EP 2007006567W WO 2008012066 A2 WO2008012066 A2 WO 2008012066A2
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
sequence
acid residues
peptide
lys
Prior art date
Application number
PCT/EP2007/006567
Other languages
English (en)
Other versions
WO2008012066A3 (fr
Inventor
Bernard P. Mahon
Mario Fares
Ashling Niruairc
Karen Scanlon
Mary O'gorman
Original Assignee
National University Of Ireland Maynooth
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National University Of Ireland Maynooth filed Critical National University Of Ireland Maynooth
Publication of WO2008012066A2 publication Critical patent/WO2008012066A2/fr
Publication of WO2008012066A3 publication Critical patent/WO2008012066A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • This invention relates to a novel peptide having antimicrobial activity.
  • B is a sequence of 1-10 amino acid residues or an amino terminal group-;
  • X is a sequence of 1-4 amino acid residues which may be the same or different, the or each amino acid residue having a pK >7 ;
  • Z is a sequence of 2-4 amino acid residues with the proviso that the first amino acid residue in the sequence and which is linked to the preceding lysine residue has a pK>7;
  • B 1 is a sequence of two amino acids, which may be the same or different, selected from lysine and/or arginine;
  • X I is a sequence of 1-15 amino acid residues or a carboxy- terminal group comprising at least one amino acid residue having a polar, uncharged side chain or a positively charged side chain at pH 7.0 (pK>7); or a pharmaceutically acceptable salt o.f the peptide of formula I.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired antimicrobial activity is retained by the peptide.
  • the amino-terminal NH 2 group and carboxy-terminal COOH group of free peptides are typically not set forth in a formula.
  • a hyphen at the amino- or caxboxy-terminus of a sequence indicates the presence of a further sequence of amino acid residues or a respective NH 2 or COOH terminal group.
  • abbreviations for amino acid residues are shown in the following Table of Correspondence:
  • B is a sequence of 1-5 amino acid residues, preferably 1 or 2 amino acid residues, which may be the same or different, selected from valine, isoleucine, leucine, alanine and glycine;
  • X is a sequence of two amino acids, which may be the same or different, selected from lysine, arginine and histidine;
  • Z is a sequence of two amino acids, which may be the same or different, selected from lysine, arginine and histidine;
  • X I is a sequence of 1-10 amino acid residues, which may be the same or different, selected from asparagine, glutamine, serine, threonine, lysine, arginine and histidine.
  • X 1 may also advantageously comprise an amino acid residue with an aliphatic side chain, such as alanine", valine, isoleucine, leucine or glycine.
  • any asparagine or glutamine residue in the peptides of the invention may be replaced by its corresponding deamidated acid " form, i.e. aspartate or glutamate, respectively.
  • the peptide of the invention has the partial sequence of human CCL28 comprising amino acid sequence 98 to 114, or an analogue thereof having antimicrobial activity, in which the natural amino acid sequence other than the cysteine residue, has been modified by substitution, insertion or deletion of amino acids.
  • the peptide of the invention comprises the following sequence of amino acid residues:
  • VaI-Cys-His-Arg-Lys-Lys-His-His-GIy-Lys-Arg-Asn-Ser-Asn-Arg- Ala-His which corresponds to amino acid sequence 98 to 114 of human CCL28.
  • the peptides of the invention may be modified at either the C-terminal or N-terminal by addition of residues to promote targeting to microbial surfaces. These modifications may include :
  • a targeting moiety such as a monoclonal antibody, or a derivative thereof such as ScFv.
  • RGD microbial Arg- Gly-Asp
  • a peptide of the invention may be obtained by recombinant means, or by chemical synthesis by any appropriate methodology such as Fmoc (Fluoroenylmethoxy carbonyl) solid phase methodologies.
  • the peptide may be obtained using a peptide synthesizer and a suitable resin such as 2- chlorotrityl chloride resin and suitable scavengers such as thioanisole, DTT and phenol, or phenol, water and triisopropylsilane, followed by purification using for example HPLC.
  • the peptides of the invention are suitable for therapeutic use against bacterial and pathogenic fungal infections of humans and animals.
  • infectious agents which may be targeted using the peptide of the invention include: Multidrug-resistant staphylococcus aureus (MRSA) infection, pathogenic Candida eg Candida albicans or fungal infections of the oral or vaginal mucosa, bacterial respiratory infections and sepsis.
  • MRSA Multidrug-resistant staphylococcus aureus
  • pathogenic Candida eg Candida albicans or fungal infections of the oral or vaginal mucosa
  • bacterial respiratory infections and sepsis bacterial respiratory infections and sepsis.
  • Other conditions which may be treated include acute bacterial exacerbation of chronic bronchitis (ABECB) caused by Streptococcus pneumoniae, Haemophilus influenzae, Haemophilus parainfluenzae, or Moraxella catarrhalis and community-acquired pneumonia due to Streptococcus pneumoniae
  • the peptides of the invention may be formulated in any vehicle suitable for in vivo, in vitro or therapeutic use.
  • the peptides may be formulated for oral or parenteral administration or they may be formulated in a cream or ointment for topical application.
  • a peptide of the invention can form the active ingredient (alone our in combi ⁇ ia.1ri ⁇ , or with a -suitable vehicle) in treatments against Pseudomonas sp.
  • Bacterial infections include those caused by E. coli, in particular enterotoxigenic E. coli, including gastroenteritis; and infections by Enterococcus sp. especially Enterococcus faecalis including vancomycin resistant strains.
  • a peptide of the invention can also be used to treat systemic fungal infections especially those caused by Candida albicans.
  • a peptide of the invention may be formulated for topical application For treating fungal infections, such as fungal skin and nail infections, caused by Candida albicans or other fungi such as Trichophyton rubrum.
  • the invention also provides a pharmaceutical composition for the treatment of microbi-al infections in humans or animals comprising a peptide according to the invention or a pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable adjuvant, carrier or diluent therefor.
  • the IC50 is significantly lower than that for existing antimicrobial peptides.
  • the peptides of the invention are based on a partial sequence of human CCL28, they avoid the proinflammatory effects associated with the full protein which would be clinically undesirable. 4.
  • the peptides of the invention unlike the complete CCL28 protein, inhibit and kill MRSA (multi-resistant staphylococcus aureus) .
  • the mechanism of action of the peptides of the invention is "membrane active" rather than specific protein active relying in part on the activity of the amidation site, therefore making the development of antimicrobial resistance more difficult. 6. They are highly- stable with an extended shelf life.
  • a peptide of the invention having the formula:
  • Arg-Ala-His-COOH (corresponding to amino acid sequence 98 to 114 of human CCL28) hereinafter referred to as "Maynosin”, was synthesised using an ABI 433A peptide synthesizer and cleaved from 2-chlorotrityl chloride resin by trifluoroacetic acid (95%) with phenol, water and triisopropylsilane as scavengers and purified by reverse phase high performance liquid chromatography.
  • Recombinant human CCL28 was obtained from Peprotech Inc., NJ 08553, USA.
  • The- antimicrobial activity of the prepared peptide (Maynosin) and human CCL2-8- was mea * sured_aga-inst the gram positive bacterium staphylococcus aureus (multi-drug sensitive- strain) , and- multi-resistant staphylococcus aureus (MRSA) .
  • the results are given in Table 1.
  • Figure 1 illustrates the antimicrobial activity of the peptide of Example 1 (Maynosin) against the gram positive bacterium Staphylococcus aureus
  • Figure 2 illustrates the antimicrobial activity of the peptide of Example 1 (Maynosin) against the multi-resistant Staphylococcus aureus (MRSA).
  • the antimicrobial activity of the peptide of Example 1 (Maynosin) against Staphyloccus aureus is compared with the published activities for three peptides designated A, B and C in Table 2.
  • Peptide A corresponds to amino acid residues 100 to 127 of human CCL 28.
  • the antimicrobial activity .for peptide A- is published in Hiemsha et al., J. Immunol 170( 3)- : 1452-61 (2003) .
  • Peptide B is known as CRAMP a ⁇ id has 38 amino acid residues in the following sequence:
  • Peptide C is known as Temporin 10a and has 10 amino acids in the following sequence: FLPLLASLFSRLL.
  • the antimicrobial activity for peptide C is published in Kim, J. B. et al. J Pept Res 58(5) : 349-56 (2001).
  • This Example demonstrates the stability of Maynosin.
  • the mechanism of action of Maynosin is not reliant on an initial tertiary structure or conformation. It is therefore highly stable, allowing, robust treatment during manufacture and an extended shelf life. This is supported by accelerated degradation studies involving storing Maynosin at 4O 0 C for 2 weeks or at 65°C for 5 minutes before incubating with bacteria. There was no appreciable loss of activity as measured by the ability to kill drug sensitive S. aureus ATCC #11632 as an indicator organism as illustrated in Figure 3 which shows the effect of S. aureus treated with Maynosin stored at 40 0 C for 2 weeks, and in Figure 4 which shows the effect of S . aureus treated with Maynosin stored at 65 0 C for 5 mins.
  • Maynosin is also resistant to multiple freeze thaw events, supporting the non reliance on an initial tertiary structure- and indicating good stability profile. There was little loss of activity as measured by the ability to kill drug sensitive S. aureus ATCC ⁇ #1163 ⁇ 2 as an indicator organism. Thris is illustrated in Figure 5 which shows the efficacy of Maynosin after repeated freeze-thaw cycles-
  • Maynosin has an extended shelf life, is amenable to economic transportation and storage, and has stability characteristics suitable for industrial manufacture.
  • Maynosin is effective over a broad pH range. Maynosin retains activity across a broad .range of salt concentrations 0-10OmM NaCl • killing the indicator organism- (S. aureus ATCC #11632) over the pH range: 6.0 to 10.0. The efficacy of Maynosin over a broad pH range is evident from the results in Table 3.
  • Maynosin is effective against a broad range of potential human and animal pathogens. Maynosin can kill pathogens associated with common infections including hospital acquired infections by Pseudomonas aeruginosa, and E. coli at low concentrations, and at moderate concentrations is effective against vancomycin resrstant Enteroco ⁇ cus faeca-lis (LD50 25 ⁇ M) . The results are shown in Table 4.
  • Maynosin is effective against a broad range of bacterial and fungal pathogens with an LD50 in the range of 14-16 ⁇ M.
  • the effectiveness of Maynosin against Candida albicans is illustrated in Figure 6.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un peptide représenté par la formule I : B-Cys-X-Lys-Lys-Z-Gly-B1X1 (I), dans laquelle B est une séquence de 1 à 10 résidus d'acides aminés ou un groupe terminal amino; X est une séquence de 1 à 4 résidus d'acides aminés qui peuvent être identiques ou différents, le ou chaque résidu d'acide aminé ayant un pK > 7; Z est une séquence de 2 à 4 résidus d'acides aminés, à la condition que le premier résidu d'acide aminé de la séquence lié au résidu lysine précédent ait un pK > 7; B1 est une séquence de deux acides aminés, qui peuvent être identiques ou différents, sélectionnés dans le groupe constitué par la lysine et/ou l'arginine; et XI est une séquence de 1 à 15 résidus d'acides aminés ou un groupe terminal carboxy contenant au moins un résidu d'acide aminé ayant une chaîne latérale polaire non chargée ou une chaîne latérale de charge positive à un pH de 7,0 (pK > 7); ou un sel de celui-ci acceptable sur le plan pharmacologique. Le peptide représenté par la formule I a une activité antimicrobienne, plus particulièrement une activité antibactérienne et antifongique.
PCT/EP2007/006567 2006-07-25 2007-07-24 Nouveau peptide WO2008012066A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IE20060548 2006-07-25
IES2006/0548 2006-07-25

Publications (2)

Publication Number Publication Date
WO2008012066A2 true WO2008012066A2 (fr) 2008-01-31
WO2008012066A3 WO2008012066A3 (fr) 2008-03-13

Family

ID=38754784

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/006567 WO2008012066A2 (fr) 2006-07-25 2007-07-24 Nouveau peptide

Country Status (1)

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WO (1) WO2008012066A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7101987B2 (en) * 1996-11-27 2006-09-05 Schering Corporation CCL27 polypeptides
US6306653B1 (en) * 1998-01-20 2001-10-23 Codon Diagnostics, Llc Detection and treatment of breast disease

Also Published As

Publication number Publication date
WO2008012066A3 (fr) 2008-03-13

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