MEDICAL DEVICES AND MATERIALS COMPRISING BIODEGRADABLE POLYESTERS

Abstract

The present invention provides a formulation comprising a biodegradable polyester compounded with a compound of Formula (I): AA-AA-AA-X-Y. The invention further provides methods of making these formulations, medical devices such as sutures comprising said formulations and methods of making said devices.

Claims

1. A formulation comprising a biodegradable polyester compounded with a compound of Formula (I):
AA-AA-AA-X-Y   (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; X 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 Y is selected from the group consisting of R.sub.1-R.sub.2-R.sub.3, R.sub.1-R.sub.2-R.sub.2-R.sub.3, R.sub.2-R.sub.2-R.sub.1-R.sub.3, R.sub.1-R.sub.3 and R.sub.4 wherein: R.sub.1 is C, O, S or N, R.sub.2 is C; each of R.sub.1 and R.sub.2 may be substituted by C.sub.1-C.sub.4 alkyl groups or unsubstituted; R.sub.3 is a group comprising 1 to 3 cyclic groups each of 5 or 6 non-hydrogen atoms, 2 or more of the cyclic groups may be fused and one or more of the cyclic groups may be substituted; R.sub.3 incorporates a maximum of 15 non-hydrogen atoms; and R.sub.4 is an aliphatic moiety having 2-20 non-hydrogen atoms, said moiety being linear branched or cyclic.

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

3. The formulation of claim 1, wherein said cationic amino acids are arginine and/or lysine.

4. The formulation of claim 1, wherein said lipophilic R group comprises 2 or more cyclic groups which may be fused or connected.

5. The formulation of claim 1, wherein said amino acid with a lipophilic R group is selected from tributyl tryptophan (Tbt) or a biphenylalanine derivative selected from Phe (4-(2-Naphthyl)), Phe (4-(1-Naphthyl)), Bip (4-n-Bu), Bip (4-Ph) or Bip (4-T-Bu).

6. The formulation of claim 1, wherein said compound is a compound of formula (II)
AA.sub.1-AA.sub.2-AA.sub.1-X-Y   (II) wherein: AA.sub.1 is a cationic amino acid; AA.sub.2 is an amino acid with a lipophilic R group, the R group having 14-27 non-hydrogen atoms; and X and Y are as defined in claim 1.

7. The formulation of claim 1, wherein said compound has the structural formula ##STR00003##

8. The formulation of claim 1, wherein said polyester is selected from the group consisting of a polylactide, polyglycolide, polydioxanone and polycaprolactone and co-polymers thereof.

9. The formulation of claim 1, wherein said polyester has an intrinsic viscosity between 0.1 and 8 dL/g.

10. The formulation of claim 1, wherein said polyester has a molecular weight between 3,000 and 30,000.

11. The formulation of claim 1, wherein said polyester is poly(D,L-lactide-co-glycolide), poly(L-lactide) or polycaprolactone.

12. The formulation of claim 1 which is a controlled release formulation.

13. The formulation of claim 1 wherein the compound of Formula (I) is releasably dispersed through the polyester.

14. A medical device comprising or consisting of a formulation of claim 1, preferably a medical device comprising said formulation as a coating thereon.

15. The device of claim 14, wherein said medical device is a surgical fastener or implant.

16. The device of claim 15, wherein said medical device is a suture.

17. A method of producing a formulation as defined in claim 1, said method comprising melting the biodegradable polyester in admixture with a compound of Formula (I).

18. A method of producing a formulation as defined in claim 1, said method comprising forming a mixture of a compound of Formula (I), a biodegradable polyester and one or more solvents capable of dissolving said compound and said polyester.

19. The method of claim 18, said method comprising (i) providing a first solution comprising a compound of Formula (I); (ii) providing a second solution comprising a biodegradable polyester, wherein said second solution is miscible with the first solution; and (iii) mixing said first and second solutions.

20. A method of producing a medical device as defined in claim 14, said method comprising (i) providing a formulation; and (ii) applying said formulation to a medical device.

21. The method of claim 20, wherein the formulation is applied by dipping the device into the formulation or painting the formulation onto the device.

22. The method of claim 20, wherein after application of the formulation to the medical device it is dried.

23. (canceled)

24. (canceled)

25. A method of treating or preventing an infection which method comprises contacting a subject in need thereof with a therapeutically effective amount of a formulation as defined in claim 1.

Description

[0109] The invention will now be further described with reference to the following non-limiting Examples and Figures, in which:

[0110] FIG. 1 is a graph showing the effect of one day topical treatment using compound 2 against Staphylococcus aureus FDA486 in a murine skin infection model. The number of colony forming units (CFU) are shown on the Y-axis and the type of topical treatment applied to the mice is shown on the X-axis. Compound 2 is also referred to herein as AMC-109.

[0111] FIG. 2 is a graph showing the effect of one day topical treatment using compound 2 against Streptococcus pyogenes in a murine skin infection model. The number of colony forming units (CFU) are shown on the Y-axis and the type of topical treatment applied to the mice is shown on the X-axis. Compound 2 is also referred to herein as AMC-109.

[0112] FIG. 3 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.

[0113] FIG. 4 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.

[0114] FIG. 5 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.

[0115] FIG. 6 is a graph showing quantitative analysis of the amount of AMC-109 released by extraction of the coated sutures. The first dataset is from the Test-01 batch, the next three datasets are three different sutures in the Test-02 batch.

[0116] FIG. 7 is a photograph showing results from the growth inhibition assay on S. aureus, first time use.

[0117] FIG. 8 is a graph showing the effect of AMC coated sutures on bacterial growth in liquid media measured by CFU in the growth media after exposure to the sutures.

[0118] FIG. 9 is a photograph showing zones of inhibition as described in Example 9.

[0119] FIG. 10 is a photograph of the wells containing inoculated TSB in which the various sutures were placed. The numbered wells are as follows: [0120] 1: S. aureus Ethibond coated with AMC-109 [0121] 2: S. aureus Ethibond non-coated [0122] 3: S. aureus growth control [0123] 4: S. epidermidis Ethibond coated with AMC-109 [0124] 5: S. epidermidis Ethibond non-coated [0125] 6: S. epidermidis growth control

[0126] FIG. 11 is a graph showing leakage (in percentage of total amount) vs time (minutes) for AMC-109 containing bioresorbable polyesters RG502 and L206S.

EXAMPLE 1

Peptide Synthesis

Chemicals

[0127] Protected amino acids Boc-Trp-OH, Boc-Arg-OH, Boc-4-phenyl-Phe and Ac-Arg-OH were purchased from Bachem AG while Boc-4-iodophenylalanine, Boc-3,3-diphenylalanine and Boc-(9-anthryl)alanine were purchased from Aldrich. Benzylamine, 2-phenylethylamine, 3-phenylpropylamine, (R)-2-phenylpropylamine, (S)-2-phenylpropylamine, N,N-methylbenzylamine, N,N-ethylbenzylamine and N,N-dibenzylamine making up the C-terminal of the peptide were purchased from Fluka except N-ethylbenzylamine which was purchased from Acros. Diisopropylethylamine (DI PEA), 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.

[0128] Preparation of Amino Acids

[0129] 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 g, 63.4 mmol) in trifluoroacetic acid (19 mL) is stirred at 70 OC 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.

[0130] 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%.

[0131] Benzylation of Boc-4-iodophenylalanine. Boc-4-iodophenylalanine (1 equivalent) was dissolved in 90% methanol in water and neutralized by addition of cesium carbonate until a weak alkaline pH (determined by litmus paper). The solvent was removed by rotary evaporation, and remaining water in the cesium 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.

[0132] 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 silicagel 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.

[0133] Preparation of Boc-Bip(n-Bu)-OBn: The title compound was prepared in 53% yield from 4-n-butylphenylboronic acid using the general procedure for Suzuki couplings. Boc-Bip(n-Bu)-OBn was isolated using an 80:20 ethyl acetate:n-hexane eluent.

[0134] Preparation of Boc-Bip(t-Bu)-OBn: The title compound was prepared in 79% yield from 4-t-butylphenylboronic acid using the general procedure for Suzuki couplings. Boc-Bip(t-Bu)-OBn was isolated using an 80:20 ethyl acetate:n-hexane eluent.

[0135] Preparation of Boc-Bip(4-Ph)-OBn: The title compound was prepared in 61% yield from 4-biphenylboronic acid using the general procedure for Suzuki couplings. Boc-Bip(4-Ph)-OBn was isolated by recrystallisation of the crude product from n-heptane.

[0136] Preparation of Boc-Bip(4-(2-Naphtyl))-OBn: The title compound was prepared in 68% yield from 2-naphtylboronic acid using the general procedure for Suzuki couplings. Boc-Bip(4-(2-Naphtyl))-OBn was isolated by recrystallisation of the crude product from n-heptane.

[0137] Preparation of Boc-Bip(4-(1-Naphtyl))-OBn: The title compound was prepared from 2-naphtylboronic acid using the general procedure for Suzuki couplings. Boc-Bip(4-(1-Naphtyl))-OBn was isolated by recrystallisation of the crude product from n-heptane.

[0138] 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.

[0139] Preparation of Boc-Bip(4-n-Bu)-OH: The title compound was prepared in 61% yield from Boc-Bip(n-Bu)-OBn using the general procedure for deesterification.

[0140] Preparation of Boc-Bip(4-t-Bu)-OH: The title compound was prepared in 65% yield from Boc-Bip(t-Bu)-OBn using the general procedure for deesterification.

[0141] Preparation of Boc-Bip(4-Ph)-OH: The title compound was prepared in 61% yield from Boc-Bip(4-ph)-OBn using the general procedure for deesterification.

[0142] Preparation of Boc-Bip(4-(2-Naphtyl))-OH: The title compound was prepared in 68% yield from Boc-Bip(4-(2-Naphtyl))-OBn using the general procedure for deesterification.

[0143] Preparation of Boc-Bip(4-(2-Naphtyl))-OH: The title compound was prepared in 68% yield from Boc-Bip(4-(2-Naphtyl))-OBn using the general procedure for deesterification.

[0144] 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.

[0145] Solution phase amide formation using PyCloP. Synthesis of Boc-Arg-N(CH.sub.2Ph).sub.2. A solution of Boc-Arg-OH(1eq), 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.

[0146] Peptide purification and analysis. The peptides were purified using reversed phase HPLC on a Delta-Pak (Waters) 018 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).

EXAMPLE 2

In Vitro Activities of Peptides Defined Herein

Materials and Methods

Antimicrobials

[0147] Vials of pre-weighed Compound 1 and Compound 2 were supplied by Lytix Biopharma AS.

TABLE-US-00001 General compound formula: AA.sub.1-AA.sub.2-AA.sub.1-XY AA.sub.1 AA.sub.2 XY Compound 1 Arg Phe(4-(2-Naphtyl)) NHCH.sub.2CH.sub.2Ph Compound 2 Arg 2,5,7-tri-tert- NHCH.sub.2CH.sub.2Ph butyltryptophan

Bacterial Isolates

[0148] 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 1. These included 54 Gram-positive bacteria, 33 Gram-negative bacteria and 10 fungi.

Determination of Minimum Inhibitory Concentration (MIC)

[0149] MICs were determined using the following microbroth dilution methods for antimicrobial susceptibility testing published by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS):

[0150] M7-A6 Vol. 23 No. 2 January 2003 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard—Sixth Edition. M100-S15 Vol. 25 No 1. January 2005 Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. M11-A6 Vol. 24 No. 2 Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard—Sixth Edition. M27-A2 Vol. 22 No. 15 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Second Edition. M38-A Vol. 22 No. 16 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard.

[0151] MIC estimations were performed using wet plates, containing the antibacterials or antifungals, prepared at GR Micro Ltd.

[0152] 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.

[0153] 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.

[0154] 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.)

[0155] 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

[0156] 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.

[0157] To evaluate whether freezing affected the activity of the peptides some MIC determinations were repeated using plates containing freshly-prepared Mueller-Hinton broth.

Control Strains

[0158] The following control (reference) strains were included in the panel of strains tested

TABLE-US-00002 Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 29213 Enterococcus faecalis ATCC 29212 Streptococcus pneumoniae ATCC 49619 Pseudomonas aeruginosa ATCC 27853 Candida krusei ATCC 6258

[0159] The control strains below were extra to the test strain panel and were included where appropriate, to check that the comparators were within range.

TABLE-US-00003 Haemophilus influenzae ATCC 49247 Candida parapsilosis ATCC 22019 Bacteroides fragilis ATCC 25285 Eggerthella lenta ATCC 43055

Results

[0160] The results are shown in Table 1 as a single line listing. Repeat control strain results are shown in Table 2. It can be seen that the control strain results were highly reproducible including data from plates that contained Mueller Hinton broth either stored frozen or used fresh. Freezing plates also had no effect on the MIC for other bacterial strains.

[0161] The MIC data obtained is very encouraging and indicates that the peptides have quite a broad spectrum of activity.

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

TABLE-US-00005 TABLE 2 In vitro activity of two antimicrobial peptides and comparators against ATCC control strains (Including ATCC control strains extra to the test strain panel) Strain Compound No. Species and properties 1 2 Plate type GP01 Staphylococcus aureus ATCC 29213 8 4 Frozen MHB antibiotic-susceptible control strain GP01 Staphylococcus aureus ATCC 29213 4 4 Frozen MHB antibiotic-susceptible control strain GP01 Staphylococcus aureus ATCC 29213 4 2 Fresh MHB antibiotic-susceptible control strain GN01 Escherichia coli ATCC 25922 32 8 Frozen MHB antibiotic-susceptible type strain GN01 Escherichia coli ATCC 25922 32 8 Frozen MHB antibiotic-susceptible type strain GN01 Escherichia coli ATCC 25922 16 8 Fresh MHB antibiotic-susceptible type strain GN10 Pseudomonas aeruginosa ATCC 27853 16 8 Frozen MHB antibiotic-susceptible type strain GN10 Pseudomonas aeruginosa ATCC 27853 32 8 Frozen MHB antibiotic-susceptible type strain GN10 Pseudomonas aeruginosa ATCC 27853 8 8 Fresh MHB antibiotic-susceptible type strain GP11 Enterococcus faecalis - ATCC 29212 8 8 Frozen MHB antibiotic-susceptible control strain GP11 Enterococcus faecalis - ATCC 29212 8 8 Frozen MHB antibiotic-susceptible control strain GP11 Enterococcus faecalis - ATCC 29212 4 4 Fresh MHB antibiotic-susceptible control strain Haemophilus influenzae - ATCC 47247 32 4 HTM Candida parapsilosis ATCC 22019 4 8 RPMI 1640 F05 Candida (Issatchenkia) krusei ATCC 6258 8 8 RPMI 1640 reference strain F05 Candida (Issatchenkia) krusei ATCC 6258 8 8 RPMI 1640 reference strain Bacteroides fragilis - ATCC 25285 64 64 SBB Eggerthella lenta - ATCC 43055 16 32 SBB
MHB, Mueller Hinton broth; HTM, haemophilus test medium; SBB, supplemented Brucella broth.

EXAMPLE 3

Stability Towards Tryptic Deciradation and Antimicrobial Activity

[0162] Compounds of formula AA.sub.1-AA.sub.2-AA.sub.1-NHCH.sub.2CH.sub.2Ph were tested for their trypsin resistance and antimicrobial activity.

Measurements and Calculation of Peptide Half-Life

[0163] Each peptide was dissolved in a 0.1 M NH.sub.4HCO.sub.3 buffer (pH 6.5) to yield a final peptide concentration of 1 mg/ml. A trypsin solution was prepared by dissolving 1 mg of trypsin in 50 ml 0.1 M NH.sub.4HCO.sub.3 buffer (pH 8.2). For the stability determination, 250 μl freshly made trypsin solution and 250 μl peptide solution were incubated in 2 ml of 0.1 M NH.sub.4HCO.sub.3 buffer (pH 8.6) at 37° C. on a rocking table. Aliquots of 0.5 ml were sampled at different time intervals, diluted with 0.5 ml water:acetonitrile (60:40 v/v) containing 1% TFA and analysed by RP-HPLC as described above. Samples without trypsin addition taken at 0 h and after 20 h at 37° C. were used as negative controls. Integration of the peak area at 254 nm for samples taken during the first 5 hours of the assay was used to generate the τ.sub.1/2. Peptides that displayed no degradation during the first 24 h were classified as stable.

Antibacterial Assay

[0164] 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.

TABLE-US-00006 TABLE 3 Stability of AA.sub.1-AA.sub.2-AA.sub.1-NHCH.sub.2CH.sub.2Ph peptides towards trypsin measured as half-life (τ.sub.1/2) and antibacterial activities displayed as MIC. MIC.sup.b (μM) Peptide AA.sub.1 AA.sub.2 τ.sub.1/2.sup.a (h) S. aureus.sup.c MRSA.sup.d MRSE.sup.e Compound 6.sup.f Arg Trp 7 145 97 81 Compound 5 Arg Bip(4-Ph) Stable 5 3 3 Compound 4 Lys 2,5,7-tri-tert- Stable 3 <2 butyltryptophan Compound 3 Arg Phe(4-(1- 20 3 3 Naphtyl)) Compound 2 Arg 2,5,7-tri-tert- Stable <3 <3 <3 butyltryptophan Compound 1 Arg Phe(4-(2- Stable 4 <3 <3 Naphtyl)) .sup.aMedical Calculator from Cornell University was used to calculate the half-life. .sup.bMinimal inhibitory concentration .sup.cStaphylococcus aures strain ATCC 25923 .sup.dMethicillin resistant Staphylococcus aureus ATCC 33591 .sup.eMethicillin resistant Staphylococcus epidermis ATCC 27626 .sup.fnot within compound definition for invention

EXAMPLE 4

In Vivo Activity of Compound 2

[0165] 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. 1 and 2, expressed as the number of colony forming units per mouse.

[0166] In experiment 1 (FIG. 1), compound 2 was applied to the murine skin as part of either a cream or a gel containing 2% (w/w) of compound 2. The same cream or gel without compound 2 was used as a negative control (placebo). It can clearly be seen that the number of CFUs was reduced when a cream or gel containing compound 2 was applied to the murine skin, compared to the negative control, indicating that compound 2 exerted an antimicrobial effect against Staphylococcus aureus. The nature of the carrier, cream or gel, had no significant effect.

[0167] In experiment 2 (FIG. 2), compound 2 was applied in two different concentrations, as either a 1% or a 2% gel. A placebo gel and a known antibacterial “bactroban” were used as controls. It can be seen that gels containing compound 2 were more effective at reducing the number of CFUs than the placebo gel or the bactroban. The gel containing 2% of compound 2 was more effective than the gel containing only 1% of compound 2.

EXAMPLE 5

Preparation and Physical, Antimicrobial and Aemolytic Properties of Compounds of Use in the Invention

[0168] Peptide Synthesis—Relevant Information is Also Provided in Example 1.

[0169] Chemicals:

[0170] 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.

[0171] 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.

[0172] 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. Boc-Phe(4-(2′-Naphtyl))-OBn was isolated by recrystallisation of the crude product from n-heptane.

[0173] 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.

[0174] 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.

[0175] General procedure for Solution phase peptide synthesis using HBTU is described in Example 1.

[0176] Solution phase amide formation using PyCloP is described in Example 1.

[0177] Peptide purification and analysis is described in Example 1.

TABLE-US-00007 TABLE 4 General compound formula: Arg-AA.sub.2-Arg-X-Y Purity Compound AA.sub.2 XY (HPLC) 7 2,5,7-tri-tert- NHCH(CH.sub.3).sub.2 butyltryptophan 8 2,5,7-tri-tert- NH(CH.sub.2).sub.5CH.sub.3 butyltryptophan 9 2,5,7-tri-tert- NH(CH.sub.2).sub.3CH.sub.3 87 butyltryptophan 10 2,5,7-tri-tert- NH(CH.sub.2).sub.2CH.sub.3 99 butyltryptophan 11 2,5,7-tri-tert- NH(CH.sub.2).sub.15CH.sub.3 80 butyltryptophan 12 2,5,7-tri-tert- NHCH.sub.2CH(CH.sub.3).sub.2 97 butyltryptophan 13 2,5,7-tri-tert- NHcyclohexyl 95 butyltryptophan 14 2,5,7-tri-tert- NHcyclopentyl 91 butyltryptophan 15 Phe(4-4′-biphenyl) NHCH(CH.sub.3).sub.2 16 Phe(4-4′-biphenyl) NH(CH.sub.2).sub.5CH.sub.3 17 Phe(4-(2′-Naphtyl)) NHCH(CH.sub.3).sub.2 18 Phe(4-(2′-Naphtyl)) NH(CH.sub.2).sub.5CH.sub.3

Antimicrobial Assay

[0178] 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.

TABLE-US-00008 TABLE 5 Antimicrobial and toxic properties of compounds of use in 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 7 25 <2 <2 <2 <2 7 7 720 8 5 2 2 <1 2 5 5 32 9 10 2 3 <2 2 350 10 10 2 3 <2 2 620 11 >100 5 4 4 6 >100 >100 38 12 10 <2 3 2 2 300 13 10 <2 2 2 <2 55 14 10 <2 >15 <2 2 340

EXAMPLE 6

In Vitro Broad Panel Screening of Selected Compounds

Materials and Methods

Antimicrobials

[0179] Vials of pre-weighed Compound 7 and Compound 8 were supplied by Lytix Biopharma AS.

TABLE-US-00009 General compound formula: AA.sub.1-AA.sub.2-AA.sub.1-X-Y AA.sub.1 AA.sub.2 XY Compound 7 Arg 2,5,7-tri-tert- NHCH(CH.sub.3).sub.2 butyltryptophan Compound 8 Arg 2,5,7-tri-tert- NH(CH.sub.2).sub.5CH.sub.3 butyltryptophan

Bacterial Isolates

[0180] Bacterial isolates used in this study are as described in Example 2.

Determination of Minimum Inhibitory Concentration (MIC)

[0181] MICs were determined as described in Example 2.

Results

[0182] The results are shown in Table 6 as a single line listing.

[0183] The MIC data obtained is very encouraging and indicates that the peptides have quite a broad spectrum of activity.

TABLE-US-00010 TABLE 6 Single line list of the in vitro activity of two antimicrobial peptides against a panel of Gram-positive bacteria, Gram-negative bacteria and fungi. Compound 7 Compound 8 Species and properties (mg/L) (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 7

In Vivo Activity of Compounds 7 and 8

[0184] 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. 3, 4 and 5 expressed as the number of colony forming units per mouse.

[0185] In experiment 1 (FIG. 3), compound 7 was applied to the murine skin as part of either a cream or a gel containing 2% (w/w) of compound 7. The same cream or gel without compound 7 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 7 was applied to the murine skin, compared to the negative control, indicating that compound 7 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.

[0186] In experiment 2 (FIG. 4), compound 7 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 7 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 7 was more effective than the gel containing only 1% of compound 7.

[0187] In experiment 3 (FIG. 5), compound 8 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 8 was more effective at reducing the number of CFUs than the placebo and fucidin or bactroban.

EXAMPLE 8

Preparation of Absorbable Braided Sutures with AMC-109 Containing Coating

[0188] The purpose was to investigate solution coating of braided sutures with a typical biodegradable poly(lactide-co-glycolide) polymer containing AMC-109. The work was in four areas: [0189] 1. Preparing a solvent (or solvent mixture) allowing dissolution of the poly(lactide-co-glycolide) polymer and AMC-109 [0190] 2. Coat “naked” braided absorbable suture material with the solution from item 1. above [0191] 3. Investigate the leakage of AMC-109 from the coated suture [0192] 4. Investigate the microbiological efficacy of the coated suture

Material and Methods

[0193] Polymer

[0194] Resomer® RG 502, Poly(D,L-Lactide-co-Glycolide) Sigma-Aldrich no. 719889 is a lactide:glycolide 50:50 mixture, ester terminated, Mw 7,000-17,000, biodegradable polymer. It is similar to biodegradable polymers generally used to coat commercial absorbable sutures. Its chemical structure is shown below:

##STR00002##

[0195] Peptide

[0196] AMC-109 is referred to herein as “Compound 2” and has the formula Arg-Tbt-Arg-NHCH.sub.2CH.sub.2Ph.

[0197] Suture

[0198] Syneture Surgilon size 4-0, a nylon braided suture with silicon coating was used in the investigations.

[0199] Procedure for the Preparation of Coated Sutures

[0200] The suture was washed with ethyl acetate to remove most of the pre-existing coating layer before coating.

[0201] Resomer® RG 502 was dissolved in ethyl acetate and AMC-109 was dissolved in ethanol. In the first test, Test-01, 50 mg of RG 502 was dissolved in 300 μl ethyl acetate and mixed with 10 mg AMC-109 dissolved in 50 μl ethanol. In the second test, Test-02, 50 mg of RG 502 was dissolved in 400 μl ethyl acetate and mixed with 10 mg AMC-109 dissolved in 110 μl ethanol. The resulting solvent mixtures were homogeneous.

[0202] The suture base material (after ethyl acetate washing) was coated by dipping repeatedly into the coating solvent mixture.

[0203] Microbiology [0204] Bacterial strain: Staphylococcus aureus [0205] Sutures used for (colony forming unit) CFU counts: [0206] Suture Surgilon 4-0 with RG502 polymer, control [0207] Suture Surgilon 4-0 with RG502 polymer/AMC-109 [0208] Suture Surgilon 4-0 (naive), control [0209] Liquid Tryptic Soy Broth (TSB) [0210] Mueller Hinton (MH) agar plates

[0211] Colonies of S. aureus were picked from a blood agar plate, and a 0.5 McFarland solution was made (1×10.sup.8 CFU), this solution was used to: [0212] 1. Inoculate MH agar plates [0213] 2. Make dilutions in TSB to obtain 1×10.sup.5 CFU

[0214] Sutures were placed on the agar plates to observe the zone of inhibition and inoculated in TSB to examine antibacterial effect of the coated suture vs. the uncoated sutures.

[0215] Samples were incubated for 16 hours at 37° C.

[0216] The cultures from tubes with sutures inoculated in TSB were used for CFU determination by serial dilutions. To investigate the long-term effect of the coated sutures, sutures were rinsed and re-inoculated with bacteria in TSB for 16 hours at 37° C.

[0217] Three parallel experiments were performed.

[0218] CFU was determined after colony counts.

Results

[0219] Preparation of Coated Sutures

[0220] Coated sutures could readily be produced by treating the sutures with a solution of Resomer RG-502 and AMC-109. The composition of the solvent mixture was critical to the ability to dissolve both Resomer RG-502 and AMC-109 and mix (and avoid phase separation or precipitation).

[0221] Quantitative Analysis of the Amount of AMC-109 Released

[0222] The coated sutures were extracted with water. The coated suture was placed in 0.4 ml water and left standing for 30 min. The sutures were removed, dried and extracted a second time, for 22 h. The extracts were analysed for the amount AMC-109 that was released by the extraction. The results are shown in FIG. 6.

Determination of the Antimicrobial Efficacy of AMC-109 Coated Sutures

[0223] The antimicrobial efficacy of the sutures coated by Resomer RG-502 and AMC-109 was assessed by an agar growth inhibition assay and a liquid broth assay.

[0224] Day 1:

[0225] A zone of inhibition was observed around the sutures coated with AMC-109, compared to a naive control suture and a suture coated with RG-502 (no AMC-109 added) (FIG. 7).

[0226] At day one there was no obvious growth in the TSB media in the tubes containing AMC-109 coated sutures. Dilutions for CFU counts were made, obtaining a CFU count of 0, 500 and 3×10.sup.3 for the AMC-109 coated sutures. In the tubes containing the controls there was visible growth, resulting in CFU counts of 5.8×10.sup.8 and 4.7×10.sup.8 (numbers are averages of the parallels) (FIG. 8).

[0227] Day 2:

[0228] At day two there was visible growth in all tubes, and there was no visible difference between tubes with Surgilon RG502/AMC and the controls.

Conclusion

[0229] Sutures can readily be coated by a solution of a biodegradable polymer and AMC-109. The resulting sutures coated with AMC have an antibacterial effect over a period of 16 hours. A final Resomer layer without AMC-109 could be added to reduce the immediate diffusion, providing a more lasting effect.

EXAMPLE 9

Sutures with AMC-109/Polymer Coating

Materials and Methods

[0230] Bacterial strains: S. aureus ATCC29213 and S. epidermidis RP42A

[0231] Sutures: Ethibond® Excel polymer suture (Johnson & Johnson) was covered with a coating of polycaprolactone+5% AMC-109. The polymer (i.e. polycaprolactone, average molecular weight (MVV) approx. 14,000) and peptide were melted by heating rapidly (over about 3 minutes) to 120° C. in a glass vial and mixing was done when the polycaprolactone was melted. The suture was dipped in the molten mixture to coat it.

[0232] Controls: uncoated Ethibond® Excel sutures

[0233] In one experiment bacterial colonies were diluted to 0.5 McFarland and spread on Mueller Hinton agar plates and in a second experiment the colonies were diluted to 0.5 McFarland and diluted 1:100 in Tryptic Soy Broth (TSB).

[0234] In the first experiment AMC coated sutures and uncoated controls were placed on inoculated plates. Plates were incubated at 37° C. for 16 hours. In the second experiment AMC coated sutures and uncoated controls were placed in inoculated media and incubated with shaking at 37° C. for 16 hours.

[0235] Results

[0236] A clear zone of inhibition was observed on the agar plates around the AMC-109 coated suture compared to control for both S. epidermidis and S. aureus (FIG. 9).

[0237] There was a clear inhibition of bacterial growth observed in wells 1 and 4 (FIG. 10) to which had been added a portion of an Ethibond suture coated with AMC.

[0238] Sutures were further stained with Syto 9 and Propidium iodide and investigated by fluorescence microscopy. There was a clear distinction between the coated and uncoated sutures; massive bacterial growth was observed on the uncoated sutures.

EXAMPLE 10

Absorbable Sutures Coated with AMC-109

[0239] The process described is scalable and suitable for industrial development.

Materials and Methods

[0240] Sutures [0241] Coviden (Medtronic) Polysorb 3-0 [0242] Ethicon Vicryl plus 3-0 (triclosan containing, positive control)

[0243] Both sutures consist of an inner braided filament of Polyglactin 910, that is covered in an outer, softer and lubricating layer consisting of a poly(D,L-lactide-co-glycolide) (lactide:glycolide 65:35) mixed with calcium stearate. The Vicryl plus suture contains additionally triclosan as an active ingredient in the outer layer.

[0244] Polymer

[0245] Rsomer (Evonik) RG-502, degradable poly(D,L-lactide-co-glycolide) (lactide:glycolide 50:50) M.sub.w 7000-17000, degradation time<3 months.

[0246] Peptide

[0247] AMC-109 as before.

[0248] Suture Stripping

[0249] The outer layer of the Polysorb sutures was (partially) removed be washing the suture with ethyl acetate for 10 min followed by water for 2 min. The sutures were dried before coating.

[0250] Suture Coating Mixture

[0251] The coating mixture was prepared by dissolving RG-502 in ethyl acetate in one vial and dissolving AMC-109 in ethanol in a second vial. The two solutions were mixed generating a slightly turbid solution that became clear upon adding 0.5 ml ethyl acetate.

[0252] Suture Coating

[0253] The Polysorb sutures (cut in pieces of 6 cm) were soaked for 10 min in the coating mixture and dried before a second soaking for 2 min in a freshly prepared coating mixture.

[0254] Two batches of sutures were prepared, one for chemical extraction analysis and zone of inhibition testing (experiment 1) and a second for efficacy test in liquid media (experiment 2).

TABLE-US-00011 TABLE 7 Masses and volumes used in the coatings. RG502 EtOAc AMC-109 EtOH Suture (mg) (ml) (mg) (ml) EtOAc Turbidity Exp. 1 15% 45.00 0.5 8 0.25 0.5 Turbid Exp. 1 5.9% 77.68 1.907 4.90 0.25 0 “Clear” Exp. 1 50.00 1.00 0 0.25 0 control Exp. 2 15% 90.00 1.5 16.1 0.5 0.5 Turbid Exp. 2 6.1% 90.00 1.5 5.9 0.5 0.5 Turbid

[0255] Extraction

[0256] A sample of sutures were selected for aqueous extraction.

[0257] First Extraction

[0258] The suture was placed in a vial and water (1 ml) was added. The samples were left for 1 h. The extraction samples were analysed by HPLC.

[0259] Second Extraction

[0260] The suture sample from the first extraction was placed in a new vial and water (1 ml) was added and extracted for 3.5 h. The second extraction samples were analysed by HPLC.

[0261] Microbiological Evaluation

[0262] Bacterial Strains: [0263] Staphylococcus aureus (8325) [0264] Pseudomonas aeruginosa (PAO1) [0265] Escherichia coli [0266] Enterococcus faecium

[0267] Overnight colonies of the different bacterial strains were used to make 0.5 McFarland (1×10.sup.8 CFU/ml) solutions in 0.5% NaCl and further diluted in tubes with 2 ml LB (Lysogeny broth) to 10.sup.5 CFU/ml. The bacterial solution was used for inoculation of agar plates for the growth zone inhibition test, and for direct inoculation of suture test samples in LB media. All samples were incubated at 37° C. for 18 hours. Enumeration of CFU was performed by making serial dilutions from i) solution in which the test samples were inoculated (LB-media), or from ii) solution (1 ml NaCl, 0.5%) with rinsed and vortexed (20 s) samples. Serial dilutions (10.sup.−1-10.sup.−6) were made in 1 ml NaCl. From the different dilutions, 100 μl was spread on blood agar plates and further incubated at 37° C. for 18 hours.

[0268] Results:

[0269] Quantification of AMC-109 Release

TABLE-US-00012 TABLE 8 Amount (μg/ml) of AMC-109 found in the aqueous suture extracts. AMC-109 First extraction Second extraction  15% 21 4 5.9% 5 NQ *NQ denotes not quantifiable

[0270] The sutures gained between 0.4 and 0.5 mg upon coating. The AMC-109 concentration in the aqueous extracts are compiled in Table 8, and the data revealed that the high loading suture liberates 25 microgram AMC-109 into the aqueous solution upon extraction for a total of 4.5 h. The low loading suture released 5 microgram AMC-109 under similar conditions.

[0271] The amounts of AMC-109 suggest that at least 30% of the AMC-109 embedded in the high loading coating is released into water within 4.5 h. The release from the low loading suture seems to be slightly lower.

[0272] Microbiological Assessment

[0273] A zone of growth inhibition was always observed around Polysorb coated with 15% AMC-RG502. In comparison, Vicryl Triclosan only showed growth inhibition of S. aureus and E. coli, Table 9.

TABLE-US-00013 TABLE 9 Zone inhibition test Inhibition zone S. aureus P. aeruginosa E. faecium E. coli Exp. 1 Polysorb 4 mm 2 mm 3 mm 3 mm 15% 3-0 Exp. 1 Polysorb 2 mm 0 mm 1 mm 0 mm 5.9% 3-0 Exp. 1 Polysorb 0 mm 0 mm 0 mm 0 mm control 3-0 Triclosan Vicryl 15 mm  0 mm 0 mm 3 mm

[0274] Direct inoculation of the suture test material in bacterial suspension resulted in bacterial growth reduction in LB media for sutures coated with 15% AMC-109 for all strains. Triclosan coated sutures inhibited growth of S. aureus only, Table 10.

TABLE-US-00014 TABLE 10 CFU from direct inoculation of suture test samples in LB media. Colony forming units (CFU) S. aureus P. aeruginosa E. faecium E. coli Exp. 2 Polysorb  0 3.4 × 10.sup.5 0 0 15% 3-0 Exp. 2 Polysorb 3.2 × 10.sup.6 1.0 × 10.sup.9 1.6 × 10.sup.3 .sup. 6 × 10.sup.8 6.1% 3-0 Control Polysorb 6.0 × 10.sup.8 1.0 × 10.sup.9 4.6 × 10.sup.7 1.5 × 10.sup.8 3-0 Triclosan Vicryl 80 1.2 × 10.sup.9 1.5 × 10.sup.7 8.0 × 10.sup.7

Conclusion

[0275] AMC-109 can be incorporated into absorbable sutures using the described solution coating technique. The coating solution is turbid suggesting a supersaturated solution. This may increase coating efficiency.

[0276] The AMC-coated sutures liberated at least 30% of their AMC-109 content into an aqueous environment within 4.5 h. The released amount of AMC-109 is dependent on the amount compounded into the suture.

[0277] AMC-109 coated sutures provide anti-colonizing efficacy against a range of Gram-positive and Gram-negative bacteria, including important pathogens where triclosan is ineffective.

EXAMPLE 11

Casting of Bioresorbable Thin Films Containing AMC-109

11.1 Bioresorbable Polymers

[0278] Resomer L206S (poly L-lactide ester terminated) (Sigma Aldrich 719854) dissolved in dichloromethane (DCM), AMC-109 dissolved in chloroform [0279] Resomer RG502 (poly-D,L-lactide-co-glycolide) (Sigma Aldrich 719889) dissolved in tetrahydrofuran (THF), AMC-109 also dissolved in THF

11.2 Preparation of Bioresorbable Thin Films

[0280] Casting Solution

[0281] The Resomer RG502 bioresorbable polymer material and AMC-109 were separately dissolved in THF in such a manner that amount of AMC-109 compared to the bioresorbable polymer in the final painting solution was 5%. The dissolution of AMC-109 in THF takes several hours.

[0282] The Resomer L2065 bioresorbable polymer material and AMC-109 were separately dissolved in dichloromethane and chloroform, respectively. The ratio between AMC-109 and L2065 was the same as above.

[0283] Casting Process

[0284] The thin film samples were prepared by placing 8 ml of the casting solution on aluminium foil with a shallow indentation or pouring the painting solution on a watch glass. After drying (several days), the thin film casting film was mechanically loosened from its surface.

11.3 Determination of AMC-109 Leakage from Bioresorbable Thin Films Containing AMC-109

[0285] Extraction

[0286] A sample was cut from the cast thin film and accurately weighed (100-150 mg), and the amount of AMC-109 in the sample was calculated. The samples were placed in vials, water (2 ml) was added and the vials shaken. Six consecutive extractions were performed. For each extraction the old extract was replaced by deionized water (2 ml). The extractions were performed with a shaking period of 10 s, 5 min, 30 min, 3 h, 22 h, and 48 h. The amount of AMC-109 in each extract was determined by UV-spectrophotometry at 280 nm using a pre-made standard curve. The results are shown graphically in FIG. 11.

11.4 Microbiological Evaluation of Bioresorbable Thin Films Containing AMC-109

[0287] Bacterial Strains: [0288] Staphylococcus aureus 8325

[0289] Modified AATCC-100 Method

[0290] Overnight colonies of S. aureus were diluted to 0.5 McFarland in 0.9% NaCl resulting in a bacterial concentration of 1×10.sup.8 bacteria. This solution was further diluted in TSB to 1×10.sup.5 bacteria.

[0291] The thin film material of L2065 was cut in pieces of approximately 0.4×0.4 cm. The material was then submerged in dH.sub.2O for 2 minutes and air dried before use. The samples were inoculated with 50 μl of the bacterial solution (1'10.sup.5). The samples were placed on a glass slide and incubated in a moisture chamber at 37° C. for 24 hours. Two biological replicates of each test material were made.

[0292] After incubation the thin film material was washed thoroughly for 2 minutes to remove AMC-109 that is readily extractable or residing directly on the surface. Then it was placed in 1000 μl NaCl and vortexed for 45 seconds, before making serial dilutions (0-10.sup.−6) and plating of 100 μl for CFU counting.

[0293] Microbiological Efficacy

[0294] Colony Forming Units

[0295] The number of CFU were below the detection limit for the AMC containing material. Compared to the control material there was a 7 log reduction in CFU numbers, see Table below.

TABLE-US-00015 TABLE CFU values for bioresorbable thin film samples containing AMC-109. TPU/polymer AMC-109 Control L206S 0 8.7 × 10.sup.7 RG502 * * *the RG502 material disintegrated and adhered strongly to the glass slide surface during the 24 h incubation period. The material could not be recovered.

11.5 Conclusion

[0296] Resomers can be dissolved by some solvents, THF and dichloromethane has the most general applicability among the solvents tested. [0297] AMC-109 can be mixed into the dissolved polymers either in THF or chloroform solution. The solvent dissolving the AMC-109 must be miscible with the solvent used to dissolve the Resomers. [0298] The resulting casting solution can be applied on several surfaces or be used for casting thin films. [0299] The casted thin films leak AMC-109, rapidly at first, sustained at a lower concentration over at least two days depending on the nature of the Resomer. [0300] The Resomer RG502 thin film shows an interesting leakage behaviour. The polymer disintegrates over time and continuously leaks AMC-109 over a prolonged period. [0301] The Resomer L206S thin film does not disintegrate as rapidly as Resomer RG502 and may have activity for a longer period of time.