WO2008012066A2

WO2008012066A2 - Novel peptide having antimicrobial activity

Info

Publication number: WO2008012066A2
Application number: PCT/EP2007/006567
Authority: WO
Inventors: Bernard P. Mahon; Mario Fares; Ashling Niruairc; Karen Scanlon; Mary O'gorman
Original assignee: National University Of Ireland Maynooth
Priority date: 2006-07-25
Filing date: 2007-07-24
Publication date: 2008-01-31
Also published as: WO2008012066A3

Abstract

This invention relates to a peptide of the formula B-Cys-X-Lys-Lys-Z-Gly-B1X1 wherein: 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; B1 is a sequence of two amino acids, which may be the same or different, selected from lysine and/or arginine; and X1 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 thereof. The formula peptide has anti-microbial activity, especially anti-bacterial and anti-fungal activity.

Description

Novel Peptide

This invention relates to a novel peptide having antimicrobial activity.

Hieshima, K., Η. Ohtani, et al. J Immunol 170(3^")^": 1452-61 (2003) have shown antimicrobial activity of human CCL28, and of a C terminal peptide thereof comprising amino acid sequence 100 to 127. However, the antimicrobial activity of these entities is relatively low and is associated with undesirable side effects.

It is an object of the invention to provide peptides having improved antimicrobial activity.

According to the invention there is provided a peptide of the formula I:

B-Cys-X-Lys-Lys-Z-Gly-B^X¹ (I)

wherein:

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; B¹ is a sequence of two amino acids, which may be the same or different, selected from lysine and/or arginine; and

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.

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, 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 NH₂ 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 NH₂ or COOH terminal group. In keeping with standard peptide nomenclature, abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

All amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus.

In a preferred embodiment of the peptide of formula I, 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; and

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. X¹ 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.

In another embodiment, 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. In a particularly preferred embodiment, 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 C-terminal peptide that is basic in character (pK>7) .

• A targeting moiety such as a monoclonal antibody, or a derivative thereof such as ScFv.

• A peptide, carbohydrate, or nucleic acid sequence corresponding to the receptor or attachment moiety on mammalian cells or tissue, eg attachment of the core peptide of any sequence which targets a microbial Arg- Gly-Asp (RGD) sequence, a microbial adhesion attachment protein/polysaccharide- ox other structure^ sialic acid, a sulphated sugar or glycoprotein, or other moiety that targets the peptide to a microbial surface.

• Chemical ^~modificatiσns such as myristoylation, acetylation" or amidation of individual amino acids may be made to incrementall-y improve-performance.

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. Examples of 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. 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 combiτia.1riσττ, or with a -suitable vehicle) in treatments against Pseudomonas sp. including urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia,, bone and joint infections, gastrointestinal infections, systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed or patients hospitalized with cancer, cystic fibrosis, or burns, or bacterial skin infections. 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.

Thus, 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 peptides of the invention have the following advantages over existing antimicrobial agents:

1. They contain an important cysteine residue (between B and X in formula I) which enhances microbial activity and amidation mediated mechanisms. They therefore represent a significant advance over existing antimicrobials .

2. The IC50 is significantly lower than that for existing antimicrobial peptides.

3. Since 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) .

5. 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.

The invention is described in the following non-limiting Examples .

Example 1

A peptide of the invention having the formula:

NH₂-VaI-Cys-His-Arg-Lys-Lys-His-His-Gly-Lys-Arg-Asn-Ser-Asn-

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. Table 1. Comparative antimicrobial activity

From Table 1 it can be seen that Maynosin has significantly higher antimicrobial activity than the entire protein CCL28 and is effective against MRSA whereas CCL28 has no measurable activity.

The results obtained for Maynosin are illustrated in Figures 1 and 2. Figure 1 illustrates the antimicrobial activity of the peptide of Example 1 (Maynosin) against the gram positive bacterium Staphylococcus aureus and 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 aτid has 38 amino acid residues in the following sequence:

ISRLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE. The antimicrobial activity for peptide B is published in Jong-Myung Ha et al. Bull. Korean Chem. Soc. 20(9): 1073 (1999).

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).

It can be seen from Table 2 that the antimicrobial activity of the peptide (Maynosin) of the invention is significantly higher than that displayed by the known peptides, in particular peptide A which is a different partial sequence of human CCL28. Example 2

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 4O⁰C 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 40⁰C for 2 weeks, and in Figure 4 which shows the effect of S . aureus treated with Maynosin stored at 65⁰C 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-

Thus, Maynosin has an extended shelf life, is amenable to economic transportation and storage, and has stability characteristics suitable for industrial manufacture.

Example 3

This Example shows that 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.

Table 3 pH of Maynosin formulation pH 6 pH 7. 0 pH 8.0 pH 9. 0 PH 10 .0

LD50 0. 38 0.22 0.32 0. 20 0- 18 (μM)

Example 4

This Example shows that 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 Enterocoσcus faeca-lis (LD50 25μM) . The results are shown in Table 4.

Table 4

Thus, Maynosin is effective against a broad range of bacterial and fungal pathogens with an LD50 in the range of 14-16 μM. The effectiveness of Maynosin against Candida albicans is illustrated in Figure 6.

Claims

1. A peptide of the formula I:

B-Cys-X-Lys-Lys-Z-Gly-B^X¹ (I)

wherein: 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; B¹ is a sequence of two amino acids, which may be the same or different, selected from lysine and/or arginine; and X¹ 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 thereof.

2. A peptide according to claim 1, wherein 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; and X¹ 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.

3. A peptide according to claim 1 or 2, wherein X¹ comprises an amino acid residue with an aliphatic side chain.

4. A peptide according to claim 3, wherein the amino acid residue with an aliphatic side chain is selected from alanine, valine, isoleucine, leucine and glycine.

5. A peptide according to any preceding claim having 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 re-s-idus, has been- modified, by substitution, insertion or deletion of amino acids.

6. A peptide according to claim 5, comprising the following sequence of amino acid residues: Val-Cys-His-Arg-Lys-Lys-His-His-Gly-Lys-Arg-Asn-Ser-Asn-Arg- Ala-His.

7. A pharmaceutical composition for the treatment of microbial infections in humans or animals comprising a peptide according to any preceding claim or a pharmaceutically acceptable salt thereof and option-ally a pharmaceutically acceptable adjuvant, carrier or diluent therefor.

8. A pharmaceutical composition according to claim 7, which is formulated for oral or parenteral administration or for topical application.

9. A pharmaceutical composition according to claim 7 or 8-, for use in the treatment of bacterial or fungal infections.

10. Use of a peptide according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating microbial infections.