Antimicrobial compounds

Abstract

The present invention relates to a compound of formula (I) AA-AA-AA-R.sub.1—R.sub.2 (I) wherein, in any order, 2 of said AA (amino acid) moieties are cationic amino acids and 1 of said AA is an amino acid with a lipophilic R group, the R group having 14-27 non-hydrogen atoms; R.sub.1 is a N atom, which may be substituted by a branched or unbranched C.sub.1-C.sub.10 alkyl or aryl group, which group may incorporate up to 2 heteroatoms selected from N, O and S; and R.sub.2 is an aliphatic moiety having 2-20 non-hydrogen atoms, said moiety being linear, branched or cyclic. The invention further relates to formulations containing these compounds, solid supports having these compounds attached thereto, the use of these compounds in therapy, particularly as antimicrobial, anti-tumour or anti-biofilm agents and non-therapeutic uses of these compounds, particularly their use in inhibiting biofilm formation or removing a biofilm.

Claims

1. A compound of formula (II):
AA.sub.1-AA.sub.2-AA.sub.3-R.sub.1—R.sub.2  (II) wherein AA.sub.1 and AA.sub.3 are independently lysine, arginine, histidine or a cationic analogue of lysine, arginine or histidine, and AA.sub.2 is tributyl tryptophan or a biphenylalanine derivative selected from the group consisting of Phe (4-(2′-naphthyl)), Phe (4-(1′-naphthyl)), Phe(4-4′-n-butylphenyl), Phe (4-4′-biphenyl) and Phe(4-4′-t-butylphenyl); R.sub.1 is a N atom, which may be substituted by a branched or unbranched C.sub.1-C.sub.10 alkyl or aryl group, which group may incorporate up to 2 heteroatoms selected from N, O and S; and R.sub.2 is an aliphatic moiety having 2-20 non-hydrogen atoms, said moiety being linear, branched or cyclic.

2. The compound of claim 1, wherein said compound is a peptide.

3. The compound of claim 1, wherein R.sub.1 is unsubstituted.

4. The compound of claim 1, wherein R.sub.2 comprises 3 to 6 non-hydrogen atoms.

5. The compound of claim 1, wherein the non-hydrogen atoms of R.sub.2 are carbon atoms.

6. The compound of claim 1, wherein R.sub.2 is a linear or branched alkyl group.

7. The compound of claim 6, wherein said alkyl group is selected from the group consisting of ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and isomers thereof, and hexyl and isomers thereof.

8. The compound of claim 1, wherein —R.sub.1-R.sub.2 together is selected from the group consisting of —NHCH(CH.sub.3).sub.2, —NH(CH.sub.2).sub.5CH.sub.3, —NH(CH.sub.2).sub.3CH.sub.3, —NH(CH.sub.2).sub.2CH.sub.3, —NHCH.sub.2CH(CH.sub.3).sub.2, —NHcycloh exyl and —NHcyclopentyl.

9. A solid support having attached thereto the compound as claimed in claim 1.

Description

(1) The invention will now be further described with reference to the following non-limiting Examples and Figures, in which

(2) FIG. 1 are graphs showing the effect of 24 h treatment with rifampicin, linezolid, tetracycline and vancomycin on 24 h old biofilm of 6 different staphylococcal strains. For each strain, bars represent from left to right: negative control, positive control, treatment with antibiotic (vancomycin, linezold, tetracycline) concentration 5 mg/L, 50 mg/L, and 500 mg/L. For rifampicin, the concentrations were 0.01 mg/L, 0.1 mg/L and 1 mg/L. Values are means of three experiments±SD. * means strong suppression of metabolic activity. ** means complete suppression of metabolic activity.

(3) FIG. 2 are graphs showing the effect of 24 h treatment with 2 different SAMPs on 24 h old biofilm of 6 different staphylococcal strains. For each strain, bars represent from left to right negative control, positive control, treatment with SAMPs in concentration 5 mg/L, 50 mg/L, and 500 mg/L. Values are means of three experiments±SD. * means strong suppression of metabolic activity. ** means complete suppression of, metabolic activity.

(4) FIG. 3 are photos showing 48 h old S. haemolyticus 51-07 biofilm grown on cover slide discs. The biofilms were stained with LIVE/DEAD staining and investigated with confocal laser scanning microscopy. Untreated biofilm (A); biofilm treated for 24 h with vancomycin50 mg/L (B); vancomycin500 mg/L (C); tetracycline 50 mg/L (D); tetracycline 500 mg/L (E); Compound 1 50 mg/L (F) and Compound 1 500 mg/L (G).

(5) FIG. 4 is a graph showing the effect of one day topical treatment against S. aureus FDA486 in a murine skin infection model. Each mouse was treated at 9 am, 12 noon and 3 pm. The skin biopsy was collected at 6 pm. The median value is shown.

(6) FIG. 5 is a graph showing the effect of one day topical treatment against Streptococcus pyogenes CS301 in a murine skin infection model. Each mouse was treated at 7 am, 10 am and 1 pm. The skin biopsy was collected at 4 pm. The median value is shown.

(7) FIG. 6 is a graph showing the effect of one day topical treatment against S. aureus FDA486 in a murine skin infection model. Each mouse was treated at 9 am, 12 noon and 3 pm. The skin biopsy was collected at 6 pm. The median value is shown.

(8) FIG. 7 is a photomicrograph of A) Uncoated aminomethylated polystyrene HL particles B) Uncoated aminomethylated polystyrene HL particles after 24 h incubation with Staphylococcus epidermidis C) Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-polystyrene particles after 24 h incubation with Staphylococcus epidermidis D) Argininyl-(2,5,7-tri-tert-butyl) tryptophanyl-argininyl-aminohexanoyl-polystyrene particles after 24 h incubation with Staphylococcus epidermidis.

EXAMPLES

Example 1

(9) Preparation and Physical, Antimicrobial and Haemolytic Properties of Molecules of the Invention

(10) Peptide Synthesis

(11) Chemicals

(12) Protected amino acids Boc-Arg-OH, and Boc-4-phenyl-Phe were purchased from Bachem AG while Boc-4-iodophenylalanine was purchased from Aldrich. isopropylamine, propylamine, hexylamine, butylamine, hexadecylamine, isobutylamine, cyclohexylamine and cyclopentylamine making up the C-terminal of the peptide were purchased from Fluka. Diisopropylethylamine (DIPEA), 1-hydroxybenzotriazole (1-HOBt), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP) and O-(benzotriazol-1-yl)-N,N,N′,N′ tetramethyluronium hexafluorophosphate (HBTU) were purchased from Fluka. 4-n-Butylphenylboronic acid, 4-t-butylphenylboronic acid, 4-biphenylboronic acid, 2-napthylboronic acid, tri ortho-tolylphosphine, benzylbromide and palladium acetate were purchased from Aldrich. Solvents were purchased from Merck, Riedel-de Haën or Aldrich.

(13) Preparation of Amino Acids

(14) Preparation of Boc-2,5,7-tri-tert-butyltryptophan-OH: A mixture of H2N-Trp-OH (1.8 g, 8.8 mmol), t-BuOH (4.7 63.4 mmol) in trifluoroacetic acid (19 mL) is stirred at 70° C. for 3 hours. The volume of the resulting mid-brown translucent solution, is reduced on a rotary evaporator at room temperature for 30 min and then triturated by means of adding 60 mL of 7% (by weight) NaHCO3 drop-wise. The gray/white granular solid obtained is then recovered by vacuum filtration and dried in vacuo at room temperature for 24 hours. The product is isolated by crystallization from a near boiling mixture of 40% ethanol in water. Volumes typically are approximately 20 mL per gram of crude product.

(15) A first crystallization from crude produces isolated product of 80-83% purity (HPLC) with respect to all other substances in the sample and approximately 94-95% purity with respect to the known TBT analogues. Yields at this stage are in the range 60-65%.

(16) Benzylation of Boc-4-iodophenylalanine. Boc-4-iodophenylalanine (1 equivalent) was dissolved in 90% methanol in water and neutralized by addition of caesium carbonate until a weak alkaline pH (determined by litmus paper). The solvent was removed by rotary evaporation, and remaining water in the caesium salt of Boc-4-iodophenylalanine was further reduced by repeated azeotropic distillation with toluene. The resulting dry salt was dissolved in dimethylformamide (DMF), benzylbromide (1.2 equivalents) was added and the resulting mixture was stirred for 6-8 h. At the end of the reaction DMF was removed under reduced pressure and an oil containing the title compound is formed. This oil was dissolved in ethyl acetate and the resulting solution was washed with equal volumes of citric acid solution (three times), sodium bicarbonate solution and brine. The title compound was isolated as a pale yellow oil in 85% yield by flash chromatography using dichloromethane:ethyl acetate (95:5) as eluent. Crystalline benzyl Boc-4-iodophenylalanine could be obtained by recrystallisation from n-heptane.

(17) General procedure for Suzuki couplings: Benzyl Boc-4-iodophenylalanine (1 equivalent), arylboronic acid (1.5 equivalents), sodium carbonate (2 equivalents), palladium acetate (0.05 equivalent) and tri ortho-tolylphosphine (0.1 equivalent) was added to a degassed mixture of dimethoxyethane (6 ml/mmol benzyl Boc-4-iodophenylalanine) and water (1 ml/mmol benzyl Boc-4-iodophenylalanine). The reaction mixture was kept under argon and heated to 80° C. for 4-6 h. After cooling to room temperature the mixture is filtered through a short pad of silica gel and sodium carbonate. The filter cake was further washed with ethyl acetate. The filtrates were combined and the solvents were removed under reduced pressure. The products were isolated by flash chromatography using mixtures of ethyl acetate and n-hexane as eluent.

(18) Preparation of Boc-Phe(4-4′-biphenyl)-OBn: The title compound was prepared in 61% yield from 4-biphenylboronic acid using the general procedure for Suzuki couplings. Boc-Phe(4-4′-biphenyl)-OBn was isolated by recrystallisation of the crude product from n-heptane.

(19) Preparation of Boc-Phe(4-(2′-Naphtyl))-OBn: The title compound was prepared in 68% yield from 2-naphtylboronic acid using the general procedure for Suzuki couplings.

(20) Boc-Phe(4-(2′-Naphtyl))-OBn was isolated by recrystallisation of the crude product from n-heptane.

(21) General procedure for deesterification of benzyl esters: The Benzyl ester is dissolved in DMF and hydrogenated for 2 days at ambient pressure using 10% Pd on carbon as catalyst. At the end of the reaction the catalyst is removed by filtration and the solvent is removed under reduced pressure. The free acids are isolated-by recrystallisation from diethyl ether.

(22) Preparation of Boc-Phe(4-4′-biphenyl)-OH: The title compound was prepared in 61% yield from Boc-Phe(4-4′-biphenyl)-OBn using the general procedure for deesterification.

(23) Preparation of Boc-Phe(4-(2′-Naphtyl))-OH: The title compound was prepared in 68% yield from Boc-Phe(4-(2-Naphtyl))-OBn using the general procedure for deesterification.

(24) General procedure for Solution phase peptide synthesis using HBTU. The peptides were prepared in solution by stepwise amino acid coupling using Boc protecting strategy according to the following general procedure. The C-terminal peptide part with a free amino group (1 eq) and the Boc protected amino acid (1.05 eq) and 1-hydroxybenzotriazole (1-HOBt) (1.8 eq) were dissolved in DMF (2-4 ml/mmol amino component) before addition of diisopropylethylamine (DIPEA) (4.8 eq). The mixture was cooled on ice and O-(benzotriazol-1-yl)-N,N,N′,N′ tetramethyluronium hexafluorophosphate (HBTU) (1.2 eq) was added. The reaction mixture was shaken at ambient temperature for 1-2 h. The reaction mixture was diluted by ethyl acetate and washed with citric acid, sodium bicarbonate and brine. The solvent was removed under vacuum and the Boc protecting group of the resulting peptide was deprotected in the dark using 95% TFA or acetylchloride in anhydrous methanol.

(25) Solution phase amide formation using PyCloP. Synthesis of Boc-Arg-N(CH.sub.2Ph).sub.2. A solution of Boc-Arg-OH (1 eq), NH(CH.sub.2Ph).sub.2 (1.1 eq) and PyCloP (1 eq) in dry DCM (filtered through alumina)(2 ml) and DMF (1 ml). The solution was cooled on ice and DIPEA (2 eq) was added under stirring. The solution was stirred for 1 h at room temperature. The reaction mixture was evaporated, and redissolved in ethyl acetate and washed with citric acid, sodium bicarbonate and brine. The solvent was removed under vacuum and the Boc protecting group of the resulting peptide was deprotected in the dark using 95% TFA.

(26) Peptide purification and analysis. The peptides were purified using reversed phase HPLC on a Delta-Pak (Waters) C.sub.18 column (100 Å, 15 μm, 25×100 mm) with a mixture of water and acetonitrile (both containing 0.1% TFA) as eluent. The peptides were analyzed by RP-HPLC using an analytical Delta-Pak (Waters) C.sub.18 column (100 Å, 5 μm, 3.9×150 mm) and positive ion electrospray mass spectrometry on a VG Quattro quadrupole mass spectrometer (VG Instruments Inc., Altringham, UK).

(27) TABLE-US-00001 TABLE 1 Compounds of the invention General compound formula: Arg-AA.sub.2-Arg-R.sub.1—R.sub.2 Purity Compound AA.sub.2 R.sub.1R.sub.2 (HPLC) 1 2,5,7-tri-tert- NHCH(CH.sub.3).sub.2 butyltryptophan 2 2,5,7-tri-tert- NH(CH.sub.2).sub.5CH.sub.3 butyltryptophan 3 2,5,7-tri-tert- NH(CH.sub.2).sub.3CH.sub.3 87 butyltryptophan 4 2,5,7-tri-tert- NH(CH.sub.2).sub.2CH.sub.3 99 butyltryptophan 5 2,5,7-tri-tert- NH(CH.sub.2).sub.15CH.sub.3 80 butyltryptophan 6 2,5,7-tri-tert- NHCH.sub.2CH(CH.sub.3).sub.2 97 butyltryptophan 7 2,5,7-tri-tert- NHcyclohexyl 95 butyltryptophan 8 2,5,7-tri-tert- NHcyclopentyl 91 butyltryptophan 9 Phe(4-4′- NHCH(CH.sub.3).sub.2 biphenyl) 10 Phe(4-4′- NH(CH.sub.2).sub.5CH.sub.3 biphenyl) 11 Phe(4-(2′- NHCH(CH.sub.3).sub.2 Naphtyl)) 12 Phe(4-(2′- NH(CH.sub.2).sub.5CH.sub.3 Naphtyl))

(28) Antimicrobial Assay

(29) MIC determinations on Staphylococcus aureus, strain ATCC 25923, Methicillin resistant Staphylococcus aureus (MRSA) strain ATCC 33591 and Methicillin resistant Staphylococcus epidermidis (MRSE) strain ATCC 27626 were performed by Toslab AS using standard methods. Amsterdam, D. (1996) Susceptibility testing of antimicrobials in liquid media, in Antibiotics in Laboratory Medicine. 4th ed (Lorian, V., Ed.) pp 75-78, Williams and Wilkins Co; Baltimore.

(30) TABLE-US-00002 TABLE 2 Antimicrobial and toxic properties of compounds of the invention C. albicans S. aureus MRSA MRSE S. pyogenes E. coli P. aeruginosa Compound (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) EC50 1 25 <2 <2 <2 <2 7 7 720 2 5 2 2 <1 2 5 5 32 3 10 2 3 <2 2 350 4 10 2 3 <2 2 620 5 >100 5 4 4 6 >100 >100 38 6 10 <2 3 2 2 300 7 10 <2 2 2 <2 55 8 10 <2 >15 <2 2 340

Example 2

(31) In Vitro Broad Panel Screening of Selected Molecules of the Invention

(32) 2.0 Materials and Methods

(33) 2.1 Antimicrobials

(34) Vials of pre-weighed Compound 1 and Compound 2 were supplied by Lytix Biopharma AS.

(35) TABLE-US-00003 General compound formula: AA.sub.1—AA.sub.2—AA.sub.1—R.sub.1R.sub.2 AA.sub.1 AA.sub.2 R.sub.1R.sub.2 Compound 1 Arg 2,5,7-tri-tert- NHCH(CH.sub.3).sub.2 butyltryptophan Compound 2 Arg 2,5,7-tri-tert- NH(CH.sub.2).sub.5CH.sub.3 butyltryptophan

(36) 2.2 Bacterial Isolates

(37) Bacterial isolates used in this study were from various sources worldwide stored at GR Micro Ltd. and maintained, with minimal sub-culture, deep frozen at −70° C. as a dense suspension in a high protein matrix of undiluted horse serum. The species used and their characteristics are listed in Table 2. These included 54 Gram-positive bacteria, 33 Gram-negative bacteria and 10 fungi.

(38) 2.3 Determination of Minimum Inhibitory Concentration (MIC)

(39) MICs were determined using the following microbroth dilution methods for antimicrobial susceptibility testing published by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS):

(40) M7-A6 Vol. 23 No. 2. January 2003 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard-Sixth Edition.

(41) M100-S15 Vol. 25 No 1. January 2005 Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement.

(42) M11-A6 Vol. 24 No. 2. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard-Sixth Edition.

(43) M27-A2 Vol. 22 No. 15. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard-Second Edition.

(44) M38-A Vol. 22 No. 16. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard.

(45) MIC estimations were performed using wet plates, containing the antibacterials or antifungals, prepared at GR Micro Ltd.

(46) Cation-adjusted Mueller-Hinton broth (Oxoid Ltd., Basingstoke, UK and Trek Diagnostic Systems Ltd., East Grinstead, UK) (supplemented with 5% laked horse blood for Streptococcus spp., Corynebacterium jeikeium and Listeria monocytogenes) was used for aerobic bacteria, with an initial inoculum of approximately 10.sup.5 colony-forming units (CFU)/mL.

(47) Haemophilus test medium (Mueller-Hinton broth containing 0.5% yeast extract and Haemophilus test medium supplement which contains 15 mg/L of each of haematin and NAD, all obtained from Oxoid Ltd., Basingstoke, UK) was used for the Haemophilus influenzae and inoculated with approximately 10.sup.5 CFU/mL.

(48) Supplemented Brucella broth (SBB) was used for the anaerobic strains with an inoculum of approximately 10.sup.6 CFU/mL. SBB is a broth consisting of 1% peptone, 0.5% ‘Lab-lemco’, 1% glucose and 0.5% sodium chloride supplemented with 5 μg/L haemin and 1 μg/L vitamin K (both obtained from Sigma Aldrich Ltd.)

(49) Yeast and filamentous fungal MIC were performed in MOPS buffered RPMI 1640 medium (MOPS buffer obtained from Sigma Aldrich Ltd., RPMI 1640 obtained from Invitrogen Ltd, Paisley, Scotland). The yeast inocula were in the range 7.5×10.sup.2-4×10.sup.3 CFU/mL and the filamentous fungi approximately 8×10.sup.3-1×10.sup.5 CFU/mL

(50) Following normal practice all the plates containing Mueller-Hinton broth were prepared in advance, frozen at −70° C. on the day of preparation and defrosted on the day of use. Fungal, Haemophilus and anaerobic MIC determinations were all performed in plates prepared on the same day.

(51) 3.0 Results

(52) The results are shown in Table 3 as a single line listing.

(53) The MIC data obtained is very encouraging and indicates that the peptides have quite a broad spectrum of activity.

(54) TABLE-US-00004 TABLE 3 Single line list of the in vitro activity of two novel antimicrobial peptides against a panel of Gram-positive bacteria, Gram-negative bacteria and fungi. Compound Compound Species and properties 1 (mg/L) 2 (mg/L) Candida albicans ATCC90028 - reference strain 32 4 Candida albicans ATCC24433 - reference strain 64 8 Candida tropicalis ATCC750 - reference strain 4 4 Candida parapsilosis ATCC90018 - reference strain 64 8 Candida (Issatchenkia) krusei ATCC6258 - reference strain 8 32 Aspergillus niger - G.R. Micro collection 32 4 Trichophyton mentagrophytes - G.R. Micro collection 8 4 Trichophyton interdigitale - G.R. Micro collection 16 4 Microsporum canis - G.R. Micro collection 16 4 Cryptococcus neoformans - G.R. Micro collection 8 2 Escherichia coli ATCC25922 - antibiotic-susceptible type strain 32 4 Escherichia coli ATCC32518 - β-lactamase positive type strain 32 8 Escherichia coli - multi-drug resistant clinical isolate 32 8 Klebsiella aerogenes NCTC11228 - antibiotic-susceptible type strain 64 8 Klebsiella aerogenes - multi-drug resistant clinical isolate 32 8 Enterobacter sp - antibiotic-susceptible clinical isolate 64 8 Enterobacter sp - multi-drug resistant clinical isolate ≥128 8 Pseudomonas aeruginosa ATCC27853 - antibiotic-susceptible type strain 32 8 Pseudomonas aeruginosa - multi-drug resistant clinical isolate 8 4 Stenotrophomonas maltophilia - antibiotic-susceptible clinical isolate 32 4 Salmonella sp - antibiotic-susceptible clinical isolate 16 8 Salmonella sp - multi-drug resistant clinical isolate 16 8 Shigella sp - antibiotic-susceptible clinical isolate 32 4 Morganella morganii - multi-drug resistant clinical isolate 32 8 Haemophilus influenzae - β-lactamase negative clinical isolate 32 16 Haemophilus influenzae - β-lactamase positive clinical isolate 16 4 Haemophilus influenzae β-lactamase negative ampicillin-resistant clinical isolate 16 8 Moraxella catarrhalis - β-lactamase positive clinical isolate 4 16 Moraxella catarrhalis - reduced fluoroquinolone susceptibility clinical isolate 8 16 Acinetobacter baumanii - antibiotic-susceptible clinical isolate 64 16 Staphylococcus aureus ATCC 29213 - antibiotic-susceptible control strain 8 4 Staphylococcus aureus ATCC 25923 - antibiotic-susceptible control strain 8 4 Staphylococcus aureus ATCC 43300 - methicillin-resistant control strain 8 4 Staphylococcus aureus - methicillin-resistant clinical isolate 8 4 Staphylococcus aureus - multi-drug-resistant clinical isolate 16 4 Staphylococcus aureus - teicoplanin-intermediate clinical isolate 16 4 Staphylococcus epidermidis antibiotic susceptible clinical isolate 4 8 Staphylococcus epidermidis methicillin-resistant clinical isolate 4 2 Staphylococcus haemolyticus - antibiotic susceptible clinical isolate 4 4 Staphylococcus saprophyticus - antibiotic susceptible clinical isolate 2 0.5 Enterococcus faecalis - ATCC 29212 antibiotic-susceptible control strain 16 4 Enterococcus faecalis vancomycin-susceptible clinical isolate 32 4 Enterococcus faecalis vancomycin-resistant (VanA) clinical isolate 32 4 Enterococcus faecalis vancomycin-resistant (VanB) clinical isolate ≥128 8 Enterococcus faecalis high-level gentamicin-resistant clinical isolate 64 8 Enterococcus faecium vancomycin-susceptible clinical isolate 16 4 Enterococcus faecium vancomycin-resistant (VanA) clinical isolate 32 4 Enterococcus faecium vancomycin-resistant (VanB) clinical isolate 16 4 Enterococcus gallinarum vancomycin-resistant (VanC) clinical isolate 8 4 Streptococcus pneumoniae - ATCC 49619 antibiotic-susceptible control strain 32 16 Streptococcus pneumoniae - penicillin-susceptible clinical isolate 32 8 Streptococcus pneumoniae - penicillin-intermediate clinical isolate 32 16 Streptococcus pneumoniae - penicillin-resistant clinical isolate 32 16 Streptococcus pneumoniae - multi-drug resistant clinical isolate 32 16 Streptococcus pyogenes - Macrolide (MLS) resistant clinical isolate 16 8 Streptococcus pyogenes - Macrolide (M-type) resistance clinical isolate 16 8 Corynebacterium jeikeium - antibiotic-susceptible clinical isolate 8 4 Corynebacterium jeikeium - multi-drug resistant clinical isolate 8 2 Listeria monocytogenes - antibiotic-susceptible clinical isolate 16 8 MU50 Staphylococcus aureus (MRSA) - VISA type strain 16 4 EMRSA3 Staphylococcus aureus (MRSA) - SSCmec type 1 8 4 EMRSA16 Staphylococcus aureus (MRSA) - SSCmec type 2 16 4 EMRSA1 Staphylococcus aureus (MRSA) - SSCmec type 3 16 4 EMRSA15 Staphylococcus aureus (MRSA) - SSCmec type 4 8 4 HT2001254 Staphylococcus aureus (MRSA) - PVL positive 8 4 Streptococcus agalactiae - antibiotic-susceptible clinical isolate 8 8 Streptococcus agalactiae - macrolide-resistant clinical isolate 16 8 Group C Streptococcus - antibiotic-susceptible clinical isolate 16 8 Group C Streptococcus - macrolide-resistant clinical isolate 32 16 Group G Streptococcus - antibiotic-susceptible clinical isolate 16 8 Group G Streptococcus - macrolide-resistant clinical isolate 16 8 Streptococcus mitis - antibiotic-susceptible clinical isolate 32 16 Streptococcus mitis - macrolide-resistant clinical isolate 64 16 Streptococcus constellatus - antibiotic-susceptible clinical isolate 64 16 Streptococcus constellatus - macrolide-resistant clinical isolate 32 16 Streptococcus oralis - antibiotic-susceptible clinical isolate 64 16 Streptococcus oralis - macrolide-resistant clinical isolate 64 16 Streptococcus bovis - antibiotic-susceptible clinical isolate 32 8 Streptococcus bovis - macrolide-resistant clinical isolate 8 2 Streptococcus sanguis - antibiotic-susceptible clinical isolate 32 16 Streptococcus sanguis - macrolide-resistant clinical isolate 32 16 Clostridium perfringens - antibiotic-susceptible clinical isolate ≥128 32 Clostridium difficile - antibiotic-susceptible clinical isolate 64 32 Propionibacterium acnes- antibiotic-susceptible clinical isolate 4 Propionibacterium acnes- antibiotic-resistant clinical isolate 2

Example 3 In Vitro Efficacy Against Biofilms

(55) Material and Methods

(56) Bacterial Strains and Growth Conditions

(57) The clinical strains used in this study are listed in Table 4.

(58) TABLE-US-00005 TABLE 4 Bacterial strains used in this study; susceptibility to antibiotics and SAMPs, and biofilm profile. Biofilm MIC antibiotics (mg/L) MIC SAMP molecules optical Strain Source RIF VAN TET LZD GEN OXA Compound 1 Compound 2 Ica.sup.d density SH.sup.a TUH Blood <0.016 4 1 0.5 64 >256 8 4 + 0.37 51-03 culture SH TUH Blood 0.016 2 0.5 0.5 64 >256 8 4 + 0.77 51-07 culture SE.sup.b TUH Blood 0.016 2 2 2 256 16 4 2 + 0.63 08-16 culture SE RP62A Blood <0.016 4 0.5 1 8 8 8 4 + 1.33 ATCC 35984 culture SA.sup.c PIA 9 Joint <0.016 2 0.5 2 1 1 8 4 + 3.20 fluid SA PIA90 Joint 0.016 2 0.5 1 0.5 1 8 2 + 0.40 fluid .sup.aSH; Staphylococcus haemolyticus .sup.bSE; Staphylococcus epidermidis .sup.cSA; Staphylococcus aureus .sup.dica; PCR detection of icaD as a marker of the operon

(59) Six staphylococcal strains (2 S. epidermidis, 2 S. haemolyticus and 2 S. aureus) were selected based on their previously known biofilm forming capacity. Bacteria were grown overnight at 37° C. in cation adjusted Mueller-Hinton II Broth (MHIIB).

(60) Antibiotics, SAMPs and Susceptibility Testing Under Planktonic Growth Condition

(61) We determined the MICs of oxacillin, gentamicin, tetracycline, vancomycin and linezolid using E-test (AB Biodisk, Solna, Sweden) and MICs of rifampicin using broth microdilution assay. Breakpoints were interpreted according to EUCAST criteria. The MIC values for Compound 1 and 2 determined with broth microdilution assay.

(62) Biofilm Formation and Quantification of Activity Against Biofilms

(63) Biofilm formation was induced in 96-well flat bottom microtitre plates (Nunclon Surface, NUNC). First, overnight cultures were diluted 1:100 in MHIIB (S. epidermidis and S. haemolyticus) or tryptic soy broth (TSB) with 5% glucose and 5% NaCl (S. aureus). 200 μl of this bacterial suspension (10.sup.7 cfu/ml) was added to each well and incubated for 24 h at 37° C. After 24 h the wells were carefully washed twice with phosphate-buffered saline (PBS) to remove planktonic bacteria. The washing procedure was carefully evaluated by measuring metabolic activity of the PBS with the Alamar blue method, described in detail below.

(64) The washed biofilms were subjected to treatment with antibiotics or SAMPs at different concentrations. Stock solutions of the tetracycline (Sigma Aldrich), vancomycin (Alpharma) and linezolid (Pfizer) were diluted in MHIIB to 5 mg/L, 50 mg/L and 500 mg/L, and rifampicin (Sigma Aldrich) was diluted in MHIIB to 0.01 mg/L, 0.1 mg/L and 1 mg/L. Trifluoroacetate salts of the SAMPs were dissolved in sterile water and diluted to 5 mg/L, 50 mg/L and 500 mg/L in MHIIB. 200 μl of antibiotics or Compound 1 or 2, in different concentrations, were added to each well and incubated for 24 h at 37° C. Positive controls were untreated biofilms only added 200 μl MHIIB. Negative controls were only 200 μl MHIIB, with no bacteria added.

(65) The metabolic activity of the biofilm was quantified with a slightly modified method previously described by Pettit et al. Antimicrob. Agents Chemother. 2005; 49: 2612-7. Briefly, after the 24 h incubation with antimicrobial agents the wells were again washed twice with PBS and then added 250 μl MHIIB with 5% Alamar blue (AB; Biosource, Camarillo, Calif., USA). AB is a redox indicator which both fluoresces and changes colour in response to chemical reduction. The extent of reduction is a reflection of bacterial cell viability. After 1 h incubation at 37° C., absorbance was recorded at 570 and 600 nm using Versamax tuneable microplate reader (Molecular Devices, Sunnyvale, Calif., USA). All assays were performed 3 times with 8 parallels. The highest and lowest value of each run was excluded from the analyses, and the remaining 18 values were averaged.

(66) The biofilm method quantifying metabolic activity was compared to a standard semiquantitative biomass-quantification method in 96-well microtitre plates. For these experiments we grew 24 h biofilms of all 6 staphylococcal strains and analyzed metabolic activity with AB, as described above. Biomass quantification on the 24 h biofilms was performed by staining the biofilm with crystal violet (CV). After staining, ethanol:acetone (70:30) was added to each well to dissolve remaining crystal violet along the walls of the wells. The optical density (OD) was then recorded at 570 nm using a spectrophotometer.

(67) Biofilm Imaging

(68) One ml aliquots of MHIIB-diluted overnight culture was used to grow S. haemolyticus TUH 51-07 biofilm on plastic coverslides (Thermanox, cellculture treated on one side, NUNC) in 24-well dishes (Falcon 3047, Becton Dickinson, N.J., USA) for 24 h. The coverslides were then washed carefully with PBS, moved to a new plate and treated for 24 h with tetracycline 50 mg/L and 500 mg/L, vancomycin 50 mg/L and 500 mg/L, or Compound 1 50 mg/L and 500 mg/L. The coverslides were washed again with 9% NaCl and stained with a LIVE/DEAD kit (Invitrogen Molecular Probes, Eugene, Oreg., USA) following the manufacturer's instructions. This stain contains SYTO 9 (green fluorescent) and propidium iodide (PI; red fluorescent), both binding to DNA. When used alone, the SYTO 9 generally stains all bacteria in a population; both those with intact and those with damaged membranes. In contrast, PI penetrates only bacteria with damaged membranes, causing a reduction in the SYTO 9 stain green fluorescence when both dyes are present. We examined treated and untreated biofilms with a Leica TCS SP5 (Leica Microsystems CMS Gmbh, Mannheim, Germany) confocal laser scanning microscope (CLSM). Images were obtained using a 63× 1.2 NA HCX PL APO water immersion lens. For detection of SYTO9 (green channel), we used the 488 nm line of the argon laser and a detection bandwidth of 495-515 nm. For PI detection (red channel), we used the 561 nm line and a detection bandwidth of 615-660 nm. The two fluorescent signals were collected sequentially at 400 Hz. Image analyses and export was performed in Leica LAS AF version 1.8.2.

(69) Statistical Analysis and Evaluations

(70) The percent reduction of AB was calculated according to the manufacturer's formula (Trek Diagnostic System). We calculated mean and standard deviations (SD) of all repeated measurements. Pearson's two-tailed correlation between the AB method and the CV method was calculated on averaged data from all 6 staphylococcal strains. Statistical analysis was performed with SPSS for Windows software version 14.0.

(71) In FIGS. 1 and 2 we present the crude percentage values of AB reduction, including positive and negative control. We defined two levels of antimicrobial suppression of metabolic activity. A strong suppression was obtained if an agent, after adjusting for the negative control, at a certain concentration caused ≥75% reduction of AB compared to positive control. A complete suppression was obtained if an agent at a certain concentration caused a reduction of AB≥negative control value+2SD.

(72) As expected, the untreated biofilm showed green cells with intact cell membrane FIG. 3a. In the biofilm subjected to treatment with Compound 1 in a concentration of 50 mg/L and especially 500 mg/L almost all cells are stained red, indicating dead bacteria (FIGS. 3f and g). In biofilm subjected to treatment with 500 mg/L tetracycline a significant part of the cells are still green indicating live bacteria with intact cell membrane (FIG. 3e). Treatment of the biofilm with vancomycin (FIG. 3d) at a concentration close to the peak values obtained in clinical practice (50 mg/L) showed predominantly green cells (live organisms).

Example 4 In Vivo Activity of Compound 1 and 2

(73) The skin of mice was infected with Staphylococcus aureus or Streptococcus pyogenes and subsequently given a total of three treatments at three hourly intervals. Three hours after the last treatment, skin biopsies were collected and the number of colony forming units (CFUs) present in the skin sample was determined. Results are shown in FIGS. 4, 5 and 6 expressed as the number of colony forming units per mouse. In experiment 1 (FIG. 4), compound 1 was applied to the murine skin as part of either a cream or a gel containing 2% (w/w) of compound 1. The same cream or gel without compound 1 was used as a negative control (placebo). Bactroban 2% cream was used as a positive control. It can clearly be seen that the number of CFUs was reduced when a cream or gel containing compound 1 was applied to the murine skin, compared to the negative control, indicating that compound 1 exerted an antimicrobial effect against Staphylococcus aureus. The efficacy of standard clinical treatment, Bactroban 2% cream, had no significant effect under the treatment regime. The nature of the carrier, cream or gel, had no significant effect.

(74) In experiment 2 (FIG. 5), compound 1 was applied in two different concentrations, as either a 1% or a 2% gel. A placebo gel and a known antibacterial “bactroban (mupericin)” were used as controls. It can be seen that gels containing compound 1 were more effective at reducing the number of CFUs from a Streptococcus pyogenes CS 301 infection than the placebo gel or the bactroban. The gel containing 2% of compound 1 was more effective than the gel containing only 1% of compound 1.

(75) In experiment 3 (FIG. 6), compound 2 was applied in a 2% cream formulation on a Staphylococcus aureus FDA 486 infection in the murine skin infection model. A placebo cream and two known antibacterials, “Fucidin (fucidic acid) ointment 2%” and “Bactroban (mupericin) cream 2%” were used as controls. It can be seen that a cream containing compound 2 was more effective at reducing the number of CFUs than the placebo and fucidin or bactroban.

Example 5

(76) Peptidic Surface Modification of Polystyrene Particles

(77) Preparation of Coated Particles

(78) 5.1

(79) To a 20 mL peptide reactor was added 560 mg (0.5 mmole) of aminomethylated polystyrene HL particles (100-200 mesh, 0.90 mmole/g substitution) which were then washed 2×10 min with 8 mL DCM. A further 8 mL of DCM was then added and the particles permitted to swell for 1 hour before the reactor was drained prior to the first coupling.

(80) 5.2

(81) To 8 mL of DMF was added 3 equivalents of Boc amino-acid and 683 mg (3.6 equivalents) of O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium (HBTU) coupling reagent. Immediately prior to transfer of the mixture to the reactor 0.855 mL (10 equivalents) of N-Ethyldiisopropylamine (DIPEA) was added and the mixture transferred to the reaction in one portion. The reactor was then agitated moderately whilst the reaction was permitted to run overnight at room temperature.

(82) 3.

(83) The particles were then washed 3×15 min with 8 mL DMF and 2×10 min with 8 mL DCM.

(84) 4.

(85) At this point a small sample was removed from the reactor and subjected to the Kaiser test in order to determine whether there was any remaining uncoupled amine.

(86) 5.

(87) If the Kaiser test gives a positive result the procedure was repeated from and including point 2 with the same amino acid. In the event that the test was negative (no uncoupled amine remaining) 8 mL of TFA/DCM (1:1) was added to the reactor to remove the Boc group from the newly coupled amino acid and the reactor agitated moderately for 1 hour.

(88) 6.

(89) The particles were then washed 3×15 min with 8 mL DCM and 2×10 min with 8 mL DMF.

(90) 7.

(91) The procedure was now repeated from and including point 2 to and including point 6 with the next amino acid to be coupled.

(92) 8.

(93) When the final amino acid unit has completed stage 5 in the procedure outlined above the particles were washed 4×15 min with 8 mL DCM and dried in the reactor under nitrogen flow for 30 min before being dried under vacuum at room temperature for 24 hours. The particles were then stored in sealed vials at 4° C.

(94) In the manner described above, the following peptide coated particles were prepared in close to quantitative yields using the appropriate amino acids:

(95) Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-polystyrene (control peptide)

(96) Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-aminohexanoyl-polystyrene (peptide according to the invention)

(97) Reduced Bacterial Colonization of Surface Coated Particles

(98) The particles prepared above were incubated with Staphylococcus epidermidis for 24 h. The amount of bacterial colonization on the bacterial surface was determined by fluorescence microscopy (excitation frequency of 485 nm, emission frequency 498 nm) after staining of the biofilm forming bacteria by Syto9 according to standard procedures.

(99) The effect on colonization was determined by visual inspection of photomicrographs of the polystyrene particles.

(100) FIG. 7 below shows the photomicrograph of:

(101) A) Uncoated aminomethylated polystyrene HL particles

(102) B) Uncoated aminomethylated polystyrene HL particles after 24 h incubation with Staphylococcus epidermidis

(103) C) Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-polystyrene particles after 24 h incubation with Staphylococcus epidermidis

(104) D) Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-aminohexanoyl-polystyrene particles after 24 h incubation with Staphylococcus epidermidis.

(105) The biofilm colonization of the polystyrene particles in FIG. 7B) can readily be observed by the fluffy, furry nature of the surface of the particles compared to the smooth surface of the polystyrene particles in FIG. 7A). FIGS. 7C) and 7D) show the effect of the two peptide coatings on the colonization by Staphylococcus epidermidis. In particular, coating with Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl (FIG. 7C)) is markedly less effective than coating with Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-aminohexanoyl (FIG. d)).

Example 6

(106) Biofilm Formation and Quantification of Activity Against Biofilms

(107) Biofilm formation was induced in 96-well flat bottom microtitre plates (Nunclon Surface, NUNC). First, overnight cultures were diluted 1:100 in MHIIB (S. epidermidis and S. haemolyticus) or tryptic soy broth (TSB) with 5% glucose and 5% NaCl (S. aureus). 200 ml of this bacterial suspension (107 cfu/ml) was added to each well and incubated for 24 h at 37° C. After 24 h the wells were carefully washed twice with phosphate-buffered saline (PBS) to remove planktonic bacteria. The washing procedure was carefully evaluated by measuring metabolic activity of the PBS with the Alamar blue method, described in detail below.

(108) The washed biofilms were subjected to treatment with the Compounds at different concentrations.

(109) Trifluoroacetate salts of the compounds were dissolved in sterile water and diluted to 5 mg/L, 50 mg/L and 500 mg/L in MHIIB. 200 μl of the Compounds, in different concentrations, were added to each well and incubated for 24 h at 37° C. Positive controls were untreated biofilms only added 200 μl MHIIB. Negative controls were only 200 μl MHIIB, with no bacteria added.

(110) The metabolic activity of the biofilm was quantified with a slightly modified method previously described by Pettit et al. Antimicrob. Agents Chemother. 2005; 49: 2612-7. Briefly, after the 24 h incubation with antimicrobial agents the wells were again washed twice with PBS and then added 250 ml MHIIB with 5% Alamar blue (AB; Biosource, Camarillo, Calif., USA). AB is a redox indicator which both fluoresces and changes colour in response to chemical reduction. The extent of reduction is a reflection of bacterial cell viability. After 1 h incubation at 37° C., absorbance was recorded at 570 and 600 nm using Versamax tuneable microplate reader (Molecular Devices, Sunnyvale, Calif., USA). All assays were performed 3 times with 8 parallels. The highest and lowest value of each run was excluded from the analyses, and the remaining 18 values were averaged.

(111) TABLE-US-00006 Peptide sequences Peptide Sequence ME 143 R-Phe(4-(2′-naphthyl))-R—NH—CH(CH3)2

(112) TABLE-US-00007 Minimum Inhibitory Concentration (MIC) on planctonic bacteria MIC ME 143 8-16 S. epidermidis 32 μg/ml 42-77 S. epidermidis 32 μg/ml 51-03 S. haemolyticus 16 μg/ml 51-07 S. haemolyticus 16 μg/ml PIA 9 S. aureus 64 μg/ml PIA 90 S. aureus 64 μg/ml

(113) TABLE-US-00008 Minimum Biofilm Inhibitory Concentration (MBIC) for ME 143 measured as survival rate (%) MIC 5 μg/ml 50 μg/ml 500 μg/ml 8-16 S. epidermidis 100 75 6 42-77 S. epidermidis 100 80 12 51-03 S. haemolyticus 100 20 9 51-07 S. haemolyticus 100 12 8 PIA 9 S. aureus 100 100 14 PIA 90 S. aureus 100 80 7