ANTIMICROBIAL FORMULATIONS COMPRISING SILICONE

Abstract

The present invention provides a controlled release formulation comprising a silicone substrate which comprises a compound of Formula (I): AA-AA-AA-X—Y. The invention further provides methods of making these formulations, medical devices such as dressings incorporating said formulations and medical uses thereof.

Claims

1-23. (canceled)

24. A formulation comprising a silicone substrate which comprises 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 C1-C10 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 R1-R2-R3, R1-R2-R2-R3, R2-R2-R1-R3, R1-R3, and R4 wherein: R1 is C, O, S or N, R2 is C; each of R1 and R2 may be substituted by C1-C4 alkyl groups or unsubstituted; R3 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; R3 incorporates a maximum of 15 non-hydrogen atoms; and R4 is an aliphatic moiety having 2-20 non-hydrogen atoms, said moiety being linear branched or cyclic; wherein the compound of formula (I) is capable of being released from the formulation.

25. The formulation of claim 24, wherein said compound is a peptide.

26. The formulation of claim 24, wherein said compound is a compound of formula (II)
AA1-AA2-AA1-X—Y  (II) wherein: AA1 is a cationic amino acid; AA2 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 24.

27. The formulation of claim 24, wherein said compound has the structural formula ##STR00003##

28. The formulation of claim 24, wherein said compound is releasably dispersed through all or a part of the silicone substrate.

29. The formulation of claim 24 wherein the silicone is medical grade silicone, and/or wherein the silicone substrate is a rubber, gel, fluid, adhesive, sealant, foam, sheet, coating or membrane.

30. A medical device comprising or consisting of a formulation of claim 24.

31. The device of claim 30, wherein said medical device is a wound dressing, indwelling device such as a catheter or valve, dermal patch, adhesive, contact lens, breast or other implant or a liner for a prosthesis.

32. The device of claim 31, wherein said adhesive is a pressure sensitive adhesive.

33. A method of producing a formulation comprising a silicone substrate compounded with a compound of formula (I) as defined in claim 24, which method comprises curing a curable silicone elastomer composition into which a compound of formula (I) has been mixed to provide the silicone substrate compounded with the compound of formula (I).

34. The method of claim 33, wherein the curable silicone elastomer composition into which a compound of formula (I) has been mixed is cured in the presence of a catalyst.

35. The method of claim 34, wherein the catalyst is a platinum catalyst.

36. The method of claim 33, wherein the curing comprises heating to a temperature in the range of 100-200° C.

37. The method of claim 33, wherein the duration of the curing is 1-5 hours.

38. The method of claim 33, wherein the compound of formula (I) has the structure ##STR00004##

39. A method of producing a formulation as defined in claim 33, said method comprising (i) preparing a solution of one or more solvents and a compound of formula (I) dissolved therein, and (ii) applying said solution to a silicone substrate.

40. The method of claim 39 wherein (A) the product of step (ii) is cured and/or dried; and/or (B) the solution comprises two solvents.

41. The method of claim 39 which method comprises dissolving a compound of formula (I) in a first solvent and then mixing it with a silicone containing formulation which also comprises a solvent which is the same as or miscible with said first solvent.

42. The method of claim 41 wherein the resultant formulation is a silicone adhesive.

43. A method of treating or preventing an infection which method comprises applying to a subject in need thereof a therapeutically effective amount of a formulation as defined in claim 24.

Description

[0151] The invention will now be further described with reference to the following non-limiting Examples and Figures, in which:

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] FIGS. 6 and 7 are collections of photographs showing the effect of AMC-109 compounded silicone on the growth of Staphylococcus epidermidis (FIG. 6) and Staphylococcus aureus (FIG. 7). Panel A (left) shows zones of inhibition around silicone pieces. Panel B (right) shows zones of inhibition after removal of the silicone pieces.

[0158] FIGS. 8 and 9 are fluorescence micrographs of sample 2/19 (left) after 16 h exposure to Staphylococcus aureus (FIG. 8) and Staphylococcus epidermidis (FIG. 9).

[0159] FIGS. 10 and 11 are collections of photographs showing the effect of AMC-109 compounded silicone on the growth of Staphylococcus aureus (FIG. 10) and Staphylococcus epidermidis (FIG. 11) after second use.

EXAMPLE 1

[0160] Peptide Synthesis

[0161] Chemicals

[0162] 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 (DIPEA), 1-hydroxybenzotriazole (1-HOBt), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP) and 0-(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.

[0163] Preparation of Amino Acids

[0164] 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° 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.

[0165] 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%.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] Preparation of Boc-Bip(4-(2-Naphtyl))-OBn: The title compound was prepared in 68% yield from 2-naphthylboronic 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.

[0172] Preparation of Boc-Bip(4-(1-Naphtyl))-OBn: The title compound was prepared from 2-naphthylboronic 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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) 018 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

[0182] In Vitro Activities of Peptides Defined Herein

[0183] Materials and Methods

[0184] Antimicrobials

[0185] 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

[0186] Bacterial Isolates

[0187] 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.

[0188] Determination of Minimum Inhibitory Concentration (MIC)

[0189] MICs were determined using the following microbroth dilution methods for antimicrobial susceptibility testing published by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS):

[0190] M7-A6 Vol. 23 No. 2 Jan. 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.

[0191] MIC estimations were performed using wet plates, containing the antibacterials or antifungals, prepared at GR Micro Ltd.

[0192] 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)/m L.

[0193] 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.

[0194] 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.) 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

[0195] 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.

[0196] To evaluate whether freezing affected the activity of the peptides some MIC determinations were repeated using plates containing freshly-prepared Mueller-Hinton broth.

[0197] Control Strains

[0198] 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

[0199] 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

[0200] Results

[0201] 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.

[0202] 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. Compound Compound Species and properties 1 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 4 2 strain 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 32 16 control 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 32 16 isolate 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

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 8 4 Frozen MHB ATCC 29213 antibiotic- susceptible control strain GP01 Staphylococcus aureus 4 4 Frozen MHB ATCC 29213 antibiotic- susceptible control strain GP01 Staphylococcus aureus 4 2 Fresh MHB ATCC 29213 antibiotic- susceptible control strain GN01 Escherichia coli 32 8 Frozen MHB ATCC 25922 antibiotic- susceptible type strain GN01 Escherichia coli 32 8 Frozen MHB ATCC 25922 antibiotic- susceptible type strain GN01 Escherichia coli 16 8 Fresh MHB ATCC 25922 antibiotic- susceptible type strain GN10 Pseudomonas aeruginosa 16 8 Frozen MHB ATCC 27853 antibiotic- susceptible type strain GN10 Pseudomonas aeruginosa 32 8 Frozen MHB ATCC 27853 antibiotic- susceptible type strain GN10 Pseudomonas aeruginosa 8 8 Fresh MHB ATCC 27853 antibiotic- susceptible type strain GP11 Enterococcus faecalis - 8 8 Frozen MHB ATCC 29212 antibiotic- susceptible control strain GP11 Enterococcus faecalis - 8 8 Frozen MHB ATCC 29212 antibiotic- susceptible control strain GP11 Enterococcus faecalis - 4 4 Fresh MHB ATCC 29212 antibiotic- susceptible control strain Haemophilus influenzae - 32 4 HTM ATCC 47247 Candida parapsilosis 4 8 RPMI 1640 ATCC 22019 F05 Candida (Issatchenkia) 8 8 RPMI 1640 krusei ATCC 6258 reference strain F05 Candida (Issatchenkia) 8 8 RPMI 1640 krusei ATCC 6258 reference strain Bacteroides fragilis - 64 64 SBB ATCC 25285 Eggerthella lenta - 16 32 SBB ATCC 43055 MHB, Mueller Hinton broth; HTM, haemophilus test medium; SBB, supplemented Brucella broth.

EXAMPLE 3 STABILITY TOWARDS TRYPTIC DEGRADATION AND ANTIMICROBIAL ACTIVITY

[0203] 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.

[0204] Measurements and Calculation of Peptide Half-Life

[0205] 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 11/2. Peptides that displayed no degradation during the first 24 h were classified as stable.

[0206] Antibacterial Assay

[0207] 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 Arg Trp  7 145 97 81 6.sup.f Compound Arg Bip(4-Ph) Stable 5 3 3 5 Compound Lys 2,5,7-tri-tert- Stable 3 <2 4 butyltryptophan Compound Arg Phe(4-(1- 20 3 3 3 Naphtyl)) Compound Arg 2,5,7-tri-tert- Stable <3 <3 <3 2 butyltryptophan Compound Arg Phe(4-(2- Stable 4 <3 <3 1 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

[0208] 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.

[0209] 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.

[0210] 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

[0211] Preparation and Physical, Antimicrobial and Haemolytic Properties of Compounds of Use in the Invention

[0212] Peptide Synthesis—relevant information is also provided in Example 1.

[0213] Chemicals:

[0214] 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.

[0215] 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.

[0216] Preparation of Boc-Phe(4-(2′-Naphtyl))-OBn: The title compound was prepared in 68% yield from 2-naphthylboronic 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.

[0217] 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.

[0218] 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.

[0219] General procedure for Solution phase peptide synthesis using HBTU is described in Example 1.

[0220] Solution phase amide formation using PyCloP is described in Example 1.

[0221] 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′- NHCH(CH.sub.3).sub.2 biphenyl) 16 Phe(4-4′- NH(CH.sub.2).sub.5CH.sub.3 biphenyl) 17 Phe(4-(2′- NHCH(CH.sub.3).sub.2 Naphtyl)) 18 Phe(4-(2′- NH(CH.sub.2).sub.5CH.sub.3 Naphtyl))

[0222] Antimicrobial Assay

[0223] 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

[0224] In Vitro Broad Panel Screening of Selected Compounds

[0225] Materials and Methods

[0226] Antimicrobials

[0227] 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

[0228] Bacterial Isolates

[0229] Bacterial isolates used in this study are as described in Example 2.

[0230] Determination of Minimum Inhibitory Concentration (MIC)

[0231] MICs were determined as described in Example 2.

[0232] Results

[0233] The results are shown in Table 6 as a single line listing.

[0234] 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 Compound Species and properties 7 (mg/L) 8 (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 16 8 isolate Moraxella catarrhalis - β -lactamase positive clinical isolate 4 16 Moraxella catarrhalis - reduced fluoroquinolone susceptibility clinical 8 16 isolate 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 32 16 strain 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

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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

[0239] 8.1 Preparation of AMC-109 Compounded Silicone Sheets

[0240] A mortar (50 mm diameter) and pestle was used to grind three 0.33 g batches of AMC-109. The resulting batches were put together and ground in the mortar together for an additional 5 minutes.

[0241] Nusil™ MED-4065 (MED-4065) is a high consistency, extrusion grade silicone elastomer that is commercially available (Avantor 0). It is supplied in two parts (Part A and Part B). Part A is a mixture comprising <1% of dodecamethylcyclohexasiloxane (CAS-No. 540-97-6). Part B is a mixture comprising <5% of Siloxanes and Silicones, dimethyl, methyl hydrogen (CAS-No. 68037-59-2) and <1% of dodecamethylcyclohexasiloxane.

[0242] 25 g of Part A and 25 g of Part B of MED-4065 were mixed on a two roll mill, gap 1 mm, resulting in Material “C”.

[0243] 19.2 g “C” and 0.8 g of the ground AMC-109 were mixed on the two roll mill, gap 0.8 mm, until visually homogenized. The homogenized mixture was then passed 10 times, gap 0.8 mm, resulting in Material “D” containing 4% w/w AMC-109.

[0244] 10 g of “D” and 10 g of “C” were mixed on the two roll mill, gap 0.8 mm, until visually homogenized. The homogenized mixture was then passed 10 times, gap 0.8 mm, resulting in Material “E” containing 2% w/w AMC-109. This dilution procedure was repeated to produce mixtures containing 1% (Material “F”) and 0.5% w/w AMC-109 (Material “G”). Each of Materials D, E, F, and G were passed 10 times, gap 0.3 mm, on the two roll mill.

[0245] From each of the materials two samples were formed using a gap of 1 mm (resulting in a sheeting of 1.5 mm thickness) and two samples were formed using of gap 0.3 mm (resulting in a sheeting of 0.5 mm thickness) respectively.

[0246] The materials were then heat cured using a platinum catalyst already present in “A” and “B” under two different curing conditions (130° C. for 4 h or 150° C. for 2 h), resulting in the samples listed in Table 7.

TABLE-US-00011 TABLE 7 Amount of Sample Curing AMC-109 Sheet no. conditions (w/w %) thickness 1/19 130° C. 4 h 4% 1.5 mm 130° C. 4 h 4% 0.5 mm 2/19 130° C. 4 h 2% 1.5 mm 130° C. 4 h 2% 0.5 mm 3/19 130° C. 4 h 1% 1.5 mm 130° C. 4 h 1% 0.5 mm 4/19 130° C. 4 h 0.5%   1.5 mm 130° C. 4 h 0.5%   0.5 mm 5/19 150° C. 2 h 4% 1.5 mm 150° C. 2 h 4% 0.5 mm 6/19 150° C. 2 h 2% 1.5 mm 150° C. 2 h 2% 0.5 mm 7/19 150° C. 2 h 1% 1.5 mm 150° C. 2 h 1% 0.5 mm 8/19 150° C. 2 h 0.5%   1.5 mm 150° C. 2 h 0.5%   0.5 mm

[0247] 8.2 Leaching of AMC-109 from Compounded Silicone Samples into Aqueous Solution

[0248] HPLC Quantitation and Standard Curve

[0249] Three analytical standards of AMC-109 for use in the quantification were prepared by dissolving 0.18 mg, 0.37 mg and 1.57 mg of AMC-109 in 5 ml of water. These were diluted prior to analysis by HPLC in the same manner as the silicone samples.

[0250] Aqueous Extraction of AMC-109 from the Compounded Silicone Samples

[0251] The compounded silicone samples were cut into samples of 500-600 mg for the 0.5 mm films and of 800-900 mg for the 1.5 mm films. The samples were further cut into 4 or 5 smaller bits before the combined cuts were placed in a vial and water (5 ml) was added. The samples were left for 40 or 42 h. A sample of 500 μl of the aqueous extract was removed and 200 μl of the HPLC solvent was added. The extraction samples were analysed by HPLC. The identity of the AMC-109 peak in the chromatograms of the compounded silicon extracts were ascertained by electrospray ionization mass spectrometry (ESI-MS).

[0252] Compounded silicone sample #1/19 (both 0.5 mm and 1.5 mm thicknesses) was selected for a second and a third extraction. Sample #1/19 was washed with water prior to the second and third extractions, which were performed as described above for the first extraction for a duration of 45 h. A sample of 500 μl was removed and 200 μl of the HPLC solvent was added. The extraction samples were analysed by HPLC.

[0253] AMC-109 was readily quantifiable by the HPLC method. The method allowed quantification over a 1 log range representing a concentration range of 40 μg/ml to 300 μg/ml. The minimum inhibitory concentration (MIC) of AMC-109 towards S. aureus and S. epidermidis is approximately 2-4 μg/ml.

[0254] The amounts of AMC-109 released from the samples in the first extraction are shown in Table 8.

TABLE-US-00012 TABLE 8 Amount (mg/5 ml) Amount of AMC-109 of AMC- in the extracts Sample 109 Curing Thick film Thin film no. (w/w %) conditions (1.5 mm) (0.5 mm) 1/19 4% 130° C. 4 h 0.56 0.70 2/19 2% 130° C. 4 h 0.20 0.31 3/19 1% 130° C. 4 h 0.08 0.13 4/19 0.5%   130° C. 4 h NQ NQ 5/19 4% 150° C. 2 h 0.67 0.95 6/19 2% 150° C. 2 h 0.24 0.30 7/19 1% 150° C. 2 h 0.11 0.16 8/19 0.5%   150° C. 2 h NQ 0.11 *NQ denotes not quantifiable

[0255] For all but the lowest compounded concentration (0.5% w/w) the concentration in the aqueous extracts were well above the MIC of the Staphylococci. The release was highest for the thinnest films, which is in accordance with a higher surface/volume ratio for the thinner films.

[0256] The amounts of AMC-109 released from Sample #1/19 in the first, second and third extractions are shown in Table 9.

TABLE-US-00013 TABLE 9 Amount (mg/5 ml) of AMC-109 in the extracts Sample Thick film Thin film no. Extraction (1.5 mm) (0.5 mm) 1/19 1st 0.56 0.70 2nd 0.22 0.33 3rd 0.13 0.27

[0257] The results from the re-extraction experiments revealed a sustained release of AMC-109 from the silicone even after the third extraction, in total representing 6 days of sustained AMC-109 release.

[0258] The aqueous conditions that the samples were exposed to in this example mimic the in vivo environment experienced by silicone implants. The results of this example therefore show silicone materials compounded with AMP-109 would be useful for preventing bacterial contamination of silicone implants in vivo.

[0259] 8.3 Microbiological Evaluation of Compounded Silicone Samples

[0260] The antimicrobial efficacy of the compounded silicone samples was assessed using an agar diffusion method.

[0261] Bacterial strains (typical Gram-positive skin bacteria): [0262] Staphylococcus aureus (ATCC29213) [0263] Staphylococcus epidermidis RP62a

[0264] Overnight colonies of S. aureus and S. epidermidis were diluted to 0.5 McFarland and spread on Mueller Hinton agar plates. AMC-109 compounded silicone samples: 1/19, 2/19, 3/19, 4/19, 5/19, 6/19, 7/19, and 8/19 (thickness either 0.5 mm or 1.5 mm) were rinsed and placed on newly inoculated plates two times (in total three times/sample). All experiments were performed in triplicates. Plates were incubated at 37° C. for 16 hours.

[0265] FIGS. 6 and 7 show the effect of AMC-109 compounded silicone on the growth of Staphylococcus epidermidis (FIG. 6) and Staphylococcus aureus (FIG. 7). Panel A (left) shows zones of inhibition around silicone pieces. Panel B (right) shows zones of inhibition after removal of the silicone pieces.

[0266] The size of the zone of inhibition of bacterial growth (measured with a ruler) is shown in Table 10.

TABLE-US-00014 TABLE 10 Amount of Sample Curing AMC-109 Sheet Zone of no. conditions (w/w %) thickness inhibition 1/19 130° C. 4 h 4% 1.5 mm 4 mm 130° C. 4 h 4% 0.5 mm 3 mm 2/19 130° C. 4 h 2% 1.5 mm 1 mm 130° C. 4 h 2% 0.5 mm 1 mm 3/19 130° C. 4 h 1% 1.5 mm 0.05 mm 130° C. 4 h 1% 0.5 mm 0.05 mm 4/19 130° C. 4 h 0.5%   1.5 mm 0.05 mm 130° C. 4 h 0.5%   0.5 mm 0.5 mm 5/19 150° C. 2 h 4% 1.5 mm 4 mm 150° C. 2 h 4% 0.5 mm 3 mm 6/19 150° C. 2 h 2% 1.5 mm 1 mm 150° C. 2 h 2% 0.5 mm 1 mm 7/19 150° C. 2 h 1% 1.5 mm 0.05 mm 150° C. 2 h 1% 0.5 mm 0.05 mm 8/19 150° C. 2 h 0.5%   1.5 mm 0.05 mm 150° C. 2 h 0.5%   0.5 mm 0.05 mm

[0267] The silicone pieces comprising the highest amount of AMC-109 (4% w/w—sample no. 1/19 and 5/19) had the highest inhibitory zone of growth in both S. epidermidis (FIG. 6) and S. aureus (FIG. 7). The inhibitory effect was dose dependent: samples 2/19 and 6/19 (2% w/w) had a zone of inhibition of 1 mm, while samples 3/19 and 7/19 (1% w/w), and 4/19 and 8/19 (0.5% w/w) had only very small zones of inhibition. The silicone pieces seemed to release AMC in a continuous manner. In FIG. 6, panel A, top picture, one can see two clear spots, one of the spots resulted from a very brief contact with a silicone piece, which was placed on the inoculated plate for one minute only.

[0268] The silicone surfaces of sample #2/19 were investigated by fluorescence microscopy after the first exposure to the bacteria on the agar plate.

[0269] The micrographs are shown in FIGS. 8 and 9. The fluorescence micrographs reveal that there is no bacterial growth on sample #2/19 neither when exposed to S. aureus (FIG. 8) nor S. epidermidis (FIG. 9).

[0270] The samples from the agar plates were rinsed and reused twice. The reused silicone gave clear, but smaller inhibition zones than during first time use as shown in FIG. 10 (S. aureus) and FIG. 11 (S. epidermidis).

[0271] Conclusions [0272] AMC-109 is liberated from the compounded silicone samples and provides anti-colonizing efficacy against typical skin bacteria, indicating suitability of antimicrobial compounded silicone materials for use as wound dressings. [0273] AMC-109 is dose-dependently released from the compounded silicone. [0274] AMC-109 compounded silicone functions as a sustained release device. [0275] The curing procedure affects the release of AMC-109 from the compounded silicon into an aqueous solution. Higher temperatures and shorter exposure times are preferred. [0276] Surprisingly, antimicrobial peptide compounded silicone elastomers can be cured at high temperatures of 130° C. or 150° C. without degrading the peptide.

EXAMPLE 9

[0277] 9.1 Preparation of AMC-109-Containing Silicone Pressure Sensitive Adhesive (PSA) and Polyester Films Coated with the AMC-109-Containing Silicone PSA

[0278] The following solutions of AMC-109 in ethanol were prepared

TABLE-US-00015 Sample no. Amount of AMC-109 (g) Amount of ethanol (ml) 83/19 0.075 7.5 84/19 0.15 7.5 85/19 0.3 7.5 86/19 0.6 7.5

[0279] Then each solution was mixed with 30 ml of silicone pressure sensitive adhesive (Nusil MED-1356, LOT: 82485, which is commercially available from Avantor and referred to herein as MED-1356) and then put on a roller mixer at room temperature. Nusil MED-1356 is a low viscosity medical grade silicone-PSA (viscosity of 250 cP [250 mPa.Math.s] measured in accordance with standards ASTM D1084 and ASTM D2196) which contains 50% of ethyl acetate solvent. Ethyl acetate and ethanol form a stable mixture.

[0280] Solution numbers 80/19, 81/19, 87/19 and 88/19 refer to the mixtures of 30 ml MED-1356 with the solutions 83/19, 84/19, 85/19 and 86/19 respectively. After 12 hours, 80/19 and 81/19 were clear liquids whereas 87/19 and 88/19 appeared turbid and inhomogeneous. 87/19 and 88/19 were cooled to 7.5° C.±2.5° C. and put on the roll mixer overnight, which provided clear and homogeneous liquids. 80/19, 81/19, 87/19 and 88/19 were kept at 7.5° C.±2.5° C. under continuous mixing on the two roll mixer for the remainder of the study. Two control samples: 30 ml MED-1356+7.5 ml ethanol (sample no. 89/19) and pure MED-1356 (sample no. 90/19) were also prepared.

[0281] Strips of polyester film (Mylar) of thickness 0.17 mm were cut to a size of 20 mm×75 mm. The cut strips were then dipped in the AMC-109-containing silicone PSA solutions and left hanging to dry at 25° C. (i.e. cured at room temperature) to provide the following samples.

TABLE-US-00016 AMC-109 Amount of weight content of Sample Solution MED-1356 Ethanol AMC-109 solid matter in the No. No. (ml) (ml) (g) PSA (%*) 91/19 80/19 30   7.5 0.075 0.5% 92/19 81/19 30   7.5 0.150 1.0% 93/19 87/19 30   7.5 0.300 2.0% 94/19 88/19 30   7.5 0.600 4.0% 95/19 89/19 30   7.5 — 0.0% 96/19 90/19 37.5 — — 0.0% *calculated assuming that the density of solid material in MED-1356 is 1 g/ml and that MED-1356 contains 50% by volume of ethyl acetate.

[0282] It was found that the AMC-109-containing silicone PSA samples were as sticky as the control samples (very sticky) and so AMC-109 can be incorporated into silicone PSA without affecting the stickiness of the silicone PSA.

[0283] 9.2 Leaching of AMC-109 from AMC-109-Containing Silicone PSA Samples into Aqueous Solution and Microbiological Evaluation

[0284] Cuttings (approximately 1 cm) of the above polyester samples coated with AMC-109-containing silicone PSA (approximately 2 cm wide) representing a surface area of 4 cm.sup.2 were placed in a vial containing 3 ml water. The cuttings were carefully shaken on an orbital shaker for 2.5 h or 24 h before samples of the aqueous extracts were collected for quantitative analysis by HPLC. The concentration of AMP-109 released into solution is shown in the table below. No additional impurity peaks were observed in the HPLC tests.

TABLE-US-00017 Extracted Extracted concentration 2.5 h concentration 24 h Sample no. AMC-109 (mg/ml) (mg/ml) 91/19 (8/20)  0.5% (EtOH) ND NQ 92/19 (9/20)    1% (EtOH) NQ 0.019  93/19 (10/20)   2% (EtOH) 0.026 0.0389 94/19 (11/20)   4% (EtOH) 0.030 0.0815 95/19 (12/20)   0% (EtOH) ND ND 96/19 (13/20) 0% ND ND *ND denotes not detected, NQ denotes not quantifiable (but detected)

[0285] For the microbiological evaluation, overnight colonies of S. epidermidis were used to make 0.5 McFarland (1×10.sup.8 CFU/ml) solutions in 0.5% NaCl and further diluted to 105 CFU/ml. 150 μl of bacterial solution was applied to the polyester samples coated with AMC-109-containing silicone PSA (0.5 mm×10 mm), placed in an incubation chamber and incubated at 37° C. for 18 hours. The material was resuspended in 5 ml NaCl, vortexed for 20 seconds and used for making serial dilutions (10.sup.−1 to 10.sup.−6) 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. The number of CFU was then assessed. All experiments were repeated two times, except for sample 92/19 the experiment was repeated four times. The results are shown in the table below. No bacteria were observed after the bacterial solution was applied to samples 92/19, 93/19 or 94/19.

TABLE-US-00018 Sample AMC-109 CFU 92/19 (9/20)  1% (EtOH) 0 93/19 (10/20) 2% (EtOH) 0 94/19 (11/20) 4% (EtOH) 0 95/19 (12/20) 0% (EtOH) 2.2 × 10.sup.7 96/19 (13/20) 0% 1.8 × 10.sup.7

[0286] Conclusions [0287] AMC-109 can be incorporated into medical grade silicone PSA using standard techniques without adversely affecting the adherent characteristics of the silicone PSA. [0288] AMC-109 is liberated from the silicon samples and provides anti-colonizing efficacy against typical skin bacteria, indicating suitability for use as an antimicrobial adhesive, for example for fixing medical devices (e.g. bandages) to skin. [0289] AMC-109 is dose-dependently released from the AMC-109-containing silicone PSA. [0290] The samples showed continued released of AMC-109 over at least 24 h. AMC-109-containing silicone PSA can therefore function as a sustained release device.

EXAMPLE 10

[0291] 10.1 Incorporation of AMC-109 into Medical Grade Silicone by a “Swell and Dry” Method

[0292] Parts A and B of MED-4065 (as described in Example 8) were mixed in a 1:1 wt. ratio and processed on a two roll mill (gap 0.8 mm), resulting in sheets of 1 mm thickness. The heat cured (1 hour at 120° C.) sheets were cut to provide silicone samples of dimensions 2×1 cm.

[0293] Swelling agents were produced by mixing:

[0294] 0.18 g AMC-109 in 2 ml ethanol and 4 ml chloroform resulting in a 2.3 wt. % solution, or

[0295] 0.09 g AMC-109 in 2 ml ethanol and 4 ml chloroform resulting in a 1.15 wt. % solution.

[0296] The silicone samples were divided into 4 pieces and soaked for various exposure times resulting in different absorptions with the aim of achieving absorption percentages of 5%, 10%, 20%, 50%, and 100%. After soaking, the samples were superficially dried with cellulose wipes, weighed, and then dried at 20° C. for 12 hours.

[0297] Sample numbers, the concentration of AMC-109 in the swelling agent, exposure time, initial weight (total of 4 pieces per sample), weight after exposure, and absorption percentage [100%×(weight after swelling−initial weight)/initial weight] are shown in the table below.

TABLE-US-00019 Concentration Weight of AMC-109 Initial after in swelling Exposure weight swelling % Sample no. agent (wt. %) time (g) (g) absorption 19/20 2.3% 10 1.072 1.122 4.7% seconds 20/20 2.3% 30 1.078 1.174 8.9% seconds 21/20 2.3% 120 1.152 1.375 19.4% seconds 22/20 2.3% 15 minutes 1.103 1.77  60.5% 25/20 2.3% 120 1.137 2.022 77.8% minutes 29/20 1.15% 10 1.064 1.113 4.6% seconds 30/20 1.15% 30 1.114 1.22  9.5% seconds 31/20 1.15% 120 1.136 1.35  18.8% seconds 32/20 1.15% 15 minutes 1.083 1.649 52.3% 28/20 1.15% 120 1.12  2.132 90.4% minutes 34/20 0.0% 120 1.108 2.173 96.1% (control)* minutes 35/20 (control)** *sample no. 35/20 is a control sample which was exposed to swelling agent not comprising AMC-109 **sample no. 35/20 is a control sample which was not exposed to swelling agent

[0298] Without wishing to be bound by theory, it is believed that during the swelling process a concentration gradient is present with the outer layer of the material being saturated whereas the inner layers are only partially saturated. By breaking off the swelling process before saturation of the whole sample is reached, a product can be obtained in which the surface layers contain the desired concentration of peptide whilst the inner layers contain less peptide. This enables more cost-effective preparation of antimicrobial articles.

[0299] 10.2 Leaching of AMC-109 into Aqueous Solution from the Silicone Samples Loaded with AMC-109 by the Swell and Dry Method and Microbiological Evaluation of these Samples

[0300] Extraction

[0301] The silicone samples prepared using the “swell and dry” method were cut into approximately equally sized parts. The cut samples were placed in a vial and water (1 ml) was added. The samples were left for 1.5 h. The extraction samples were then filtered and analysed by HPLC. The results are shown in the table below.

TABLE-US-00020 AMC-109 concentration Concentration in in swelling agent aqueous extract Sample No. (wt. %) Swell time (mg/ml) 19/20 2.3%  10 seconds 0.023 20/20 2.3%  30 seconds 0.011 21/20 2.3% 120 seconds 0.012 22/20 2.3%  15 minutes 0.024 25/20 2.3% 120 minutes 0.049 29/20 1.15%  10 seconds 0.005 30/20 1.15%  30 seconds 0.007 31/20 1.15% 120 seconds 0.009 32/20 1.15%  15 minutes 0.11  28/20 1.15% 120 minutes 0.014 34/20 (control) 0.0% 120 minutes ND 35/20 (control) No swelling ND

[0302] Microbiological Assay

[0303] Overnight colonies of S. epidermidis (RP62A) were diluted to 0.5 McFarland (1×10.sup.8 CFU/ml) in 0.85% NaCl. The bacterial solution was further diluted in Tryptic Soy Broth media (to 1×10.sup.5 CFU/ml) and drops of 100 μl were applied to the surfaces of the silicone samples. The silicone samples were placed on glass microscope slides, which were then placed in a moist incubation chamber and incubated for 24 hours at 37° C.

[0304] For determining CFU values, the samples were vortexed in 2 ml 0.85% NaCl and serial dilutions (10.sup.−1 to 10.sup.−6) were made. 100 μl aliquots of the serial dilutions were streaked on blood agar plates, and further incubated overnight, prior to CFU counting. All experiments were performed twice. The results are shown in the table below.

TABLE-US-00021 Swelling agent AMC-1109 No. of CFU Sample no. content Exposure time (CFU/ml) 21/20 2.3% 120 s 0 22/20 2.3% 15 min 0 25/20 2.3% 120 min 0 29/20 1.15% 10 s 0 30/20 1.15% 30 s 0 31/20 1.15% 120 s 0 32/20 1.15% 15 min 0 28/20 1.15% 120 min 0 34/20 Control 0.0% 10 s 3.3 × 10.sup.8 35/20 Control No swelling —   9 × 10.sup.8

[0305] The level of bacterial inhibition was examined by number of CFU. The material showed good antimicrobial effect. All control samples showed a high level of bacterial surface colonisation.

[0306] Conclusions [0307] AMC-109 can be reversibly loaded into medical grade silicon using a swell and dry method. [0308] AMC-109 will be liberated from the samples and provide anti-colonizing and antimicrobial activity in the local environment around the silicone article as well as on the surface and interior of the silicone article. [0309] The released amount of AMC-109 is generally dependent on the amount of active ingredient in the swelling solvent and the swell time

EXAMPLE 11

[0310] 11.1 Incorporation of AMC-109 into a Commercially Available Silicone Prosthetic Liner by a “Swell and Dry” Method

[0311] Samples were cut using a scalpel from a silicone prosthetic liner (Iceross® Original Locking under limb prosthesis) that is commercially available from the Icelandic company Össur. The samples were cut into rectangular pieces (2 cm×1 cm) and had a thickness of approximately 2 mm. The swelling was performed using a mixture containing 100 mg of AMC-109 in 4 ml of 2-propanol and 8 ml of pentane. The dissolution of AMC-109 in 2-propanol is rather slow and 2-3 h is required to obtain a clear mixture. Three swell times were used, 5 min, 15 min and 30 min. To correct for AMC-109 deposited on the surface, and not penetrated into the material, a series of samples were quickly washed with water before subsequent analysis. The table below shows the sample number, swell time and an indication of whether the sample was quickly washed with water before subsequent analysis.

TABLE-US-00022 Sample no. Swell time Aqueous washing WS-070  5 minutes Yes WS-072  5 minutes No WS-074 15 minutes Yes WS-069 30 minutes Yes WS-071 30 minutes No

[0312] 11.2 Leaching of AMC-109 into Aqueous Solution from the Silicone Samples

[0313] One sample of each preparation was analysed with respect to aqueous extraction from the specimen. The silicone samples were cut in two approximately equally sized parts that were placed in a vial and water (1 ml) was added. The samples were left for 0.5 hours. The extraction samples were filtered and analysed by HPLC using UV-detection at 280 nm and quantified using a standard curve. The table below shows the concentration of AMC-109 in the extracts.

TABLE-US-00023 Sample no. Swell time Aqueous washing Concentration (mg/ml) WS-070  5 minutes Yes 0.03 WS-072  5 minutes No 0.29 WS-074 15 minutes Yes 0.05 WS-069 30 minutes Yes 0.11 WS-071 30 minutes No 0.53

[0314] The results from the aqueous extraction shows a clear correlation between the swell time and the amount of extractable AMC-109. The results also show that a relatively large amount of AMC-109 resides on the surface, and is readily removable by a quick water wash, but also that a sizable amount is absorbed by the silicone material. The amount of absorbed, but extractable AMC-109 would provide an effective antimicrobial environment in the vicinity of the product.

[0315] 11.3 Microbiological Evaluation

[0316] Bacterial Strains: [0317] Staphylococcus epidermidis RP62A [0318] Staphylococcus hominis 58-69

[0319] Overnight colonies of S. epidermidis or S. hominis were diluted to 0.5 McFarland (1×10.sup.8 CFU/ml) in 0.85% NaCl. The bacterial solution was further diluted in Tryptic Soy Broth media (to 1×10.sup.5 CFU/ml) and drops of 100 μl was applied to the surfaces of the silicone patches. Silicone patches were placed on glass microscope slides, and the glass slides were placed in a moist incubation chamber and incubated for 24 hours at 37° C.

[0320] For determining CFU, the samples were vortexed in 1 ml 0.85% NaCl and serial dilutions (10.sup.−1 to 10.sup.−6) were made. 100 μl aliquots of the serial dilutions were streaked on blood agar plates, and further incubated overnight prior to CFU counting.

[0321] The level of bacterial inhibition was examined by number of CFU and growth zone inhibition. All materials tested were washed with water after completion of the swell and dry procedure to ensure that the activity of the materials was due to internally absorbed AMC-109 only.

[0322] The table below shows the swelling time and number of CFU for bacteria grown on the silicone surfaces. Silicone swollen for both 5 and 15 minutes showed excellent antimicrobial efficiency compared to control samples (silicone swollen without AMC-109).

TABLE-US-00024 5 minutes swell 15 minutes swell time time Control Bacterial strain CFU/ml CFU/ml CFU/ml S. epidermidis 0 0 1.4 × 10.sup.8 S. hominis 0 0 1.8 × 10.sup.7

[0323] The table below shows the size of the growth inhibition zones around the silicone surfaces. The growth inhibition zones did not differ between the two samples with different swelling times in which the swelling agent contained AMC-109. The control samples (silicone swollen without AMC-109) showed no growth zone inhibition

TABLE-US-00025 5 minutes swell 15 minutes swell Bacterial strain time time Control S. epidermidis 3 mm 3 mm 0 S. hominis 4 mm 2 mm 0

[0324] Conclusions [0325] AMC-109 can be compounded into silicon prosthetic liners using a swell and dry method. [0326] A swelling agent comprising a 2-propanol/pentane solvent mixture is suitable for providing reversible swelling. Reversible swelling is desirable to avoid deformation of the impregnated material but still give deposition of the active agent within the material. [0327] AMC-109 was liberated from the samples and provided anti-colonizing and local antimicrobial efficacy against both S. aureus and S. hominis (the latter responsible for much of the unpleasant smell associated with silicone liners). [0328] The released amount of AMC-109 is dependent on the swell time.

EXAMPLE 12

[0329] 12.1 Incorporation of AMC-109 into Silicone Foam Wound Dressing Adsorbents

[0330] Preparation of AMC-109 Containing Silicone Wound Dressings

[0331] Material: Mepilex Lite (Mölnlycke Healthcare) [0332] Allevyn Gentle border (Smith & Nephew)

[0333] The silicone foam absorbent from commercial dressings were cut into 2 cm×2 cm square patches using scissors. Ethanolic solutions (400 μl) with varying amounts of AMC-109 were added dropwise to evenly cover the surface of the cut patches before drying in a fume hood for 72 hours. Control samples of the absorbent dressings were made similarly using ethanol only.

[0334] 12.2 Microbiological Analysis

[0335] Modified AA TCC 100 Method

[0336] Overnight colonies of S. aureus were diluted to 0.5 McFarland (1×10.sup.8 CFU/ml) in 0.9% NaCl and further diluted to 10.sup.5 CFU/ml in Tryptic Soy Broth media (TSB). The different silicone samples were inoculated with 400 μl of the bacterial solution and incubated at 37° C. for 24 hours in an incubation chamber. After incubation the material was vortexed in 4 ml 0.9% NaCl for 20 seconds and used for making serial dilutions (10.sup.−1-10.sup.−6) in 0.9% NaCl. From the different dilutions, 100 μl was spread on tryptic soy agar plates and further incubated at 37° C. for 24 hours. Three biological replicates of each test material were made.

[0337] Results

[0338] Colony forming counts for S. aureus (median data shown).

TABLE-US-00026 Colony forming units 0.05% AMC- 0.2% AMC- 0.4% AMC- 0% AMC-109 109 109 109 Mepilex Lite 4.5 × 10.sup.8 3.5 × 10.sup.2 0 0 Allevyn gentle 1.9 × 10.sup.8 0 0 0 border

[0339] Conclusions [0340] Silicone foam can be impregnated with AMC-109 [0341] AMC-109 was liberated from the samples and provided antimicrobial efficacy against S. aureus.

EXAMPLE 13

[0342] 13.1 Incorporation of AMC-109 into Solid Silicone by a “Swell and Dry” Method

[0343] Silicone Membrane Samples: Cylindrical pieces (8 mm diameter, 6 mm height) composed of medical grade silicone (solid silicone membrane valves produced by Tada Medical AB).

[0344] Swelling & Drying

[0345] The swelling solvent used was a 2:1 (v) mixture of 2-propanol and pentane. Four rounds of swelling were performed: [0346] 1. High loading—Silicone valves submerged in swelling solvent for 30 min with and without AMC-109 (1 mg/0.120 ml) [0347] 2. Low loading—Silicone valves submerged in swelling solvent for 30 min with and without AMC-109 (0.2 mg/0.120 ml) [0348] 3. Low loading with washing—Silicone valves submerged in swelling solvent for 30 min with AMC-109 (0.375 mg/0.120 ml). The valves were dried (1 h) and washed (dipped in 2-propanol) and dried (48 h). Control material was treated analogously in swelling solvent without AMC-109. [0349] 4. Low loading with expanded bacterial panel—Silicone valves submerged in swelling solvent for 30 min with and without AMC-109 (0.2 mg/0.120 ml). Tested against S. epidermidis (RP62a), E. coli (5067-6002) and P. aeruginosa (PAO1).

[0350] 13.2 Microbiological Assay

[0351] Modified AA TCC 100 Method

[0352] Overnight colonies of S. aureus, S. epidermidis, E. coli and P. aeruginosa were diluted to 0.5 McFarland in 0.85% 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.

[0353] After being compounded with AMC-109 by the swell and dry method, TADA silicone material was cut in pieces of 0.4 cm, the diameter of the piece was 0.7 cm, the pieces were then vertically sliced in two halves. Both sliced and unsliced samples were tested. The different samples were inoculated with 50 μl of the bacterial solution (1×10.sup.5) and incubated at 37° C. for 24 hours. Bacterial solution was applied to an original surface of the material or onto a surface created by slicing the material. Three biological replicates of each test material were made.

[0354] After incubation the silicone material was placed in 500 μl NaCl and vortexed for 20 seconds, before making serial dilutions (0-10.sup.−6) and plating of 100 μl for CFU counting.

[0355] 13.3 Results

[0356] Swelling Efficacy

[0357] The swelling was performed as described and the material swelled to a substantial degree (by visual assessment—the base of the valve increased in diameter by 10-20%). This linear swelling of approximately 15% represents a volume increase of approximately 50%.

[0358] After drying the silicone membrane samples regained its original size (by visual inspection).

[0359] Microbiological Efficacy

[0360] The number of CFU was zero for all material containing AMC-109., compared to the control material where the CFU was determined to be in the range of 10.sup.4 to 10.sup.8, as shown in the table below.

[0361] S. aureus (ATCC 8325) CFU values for silicone membrane samples impregnated with AMC-109.

TABLE-US-00027 Surface CFU inoculated (samples AMC-109 Swell with bacterial containing CFU loading concentration solution AMC-109) (Control) High 8.3 mg/ml A* 0 2.53 × 10.sup.8  Low with 3.1 mg/ml A* 0  2.7 × 10.sup.6  washing Low with 3.1 mg/ml B* 0  8.3 × 10.sup.4  washing Low 1.7 mg/ml B* 0  4.7 × 10.sup.5, A* = an original surface of the material B* = a surface created by slicing the material

[0362] S. epidermidis (RP62a), E. coli (5067-6002) and P. aeruginosa (PAO1) CFU values for silicone membrane samples impregnated with AMC-109 (low loading).

TABLE-US-00028 Surface inoculated with CFU (samples bacterial containing AMC- solution 109) CFU (Control) P. aeruginosa A* 0 2.8 × 10.sup.8 E. coli A* 0 9.6 × 10.sup.7 S. epidermidis A* 0 3.5 × 10.sup.8 A* = an original surface of the material

[0363] All material swelled in the presence of AMC-109, showed no colonization and complete bacterial killing. Two approaches were used to eliminate the AMC-109 deposited on the surface of the material (i.e. not truly integrated); either washing with 2-propanol or by cutting and exposing the inner surface only. Either technique, either alone or in concert, proved complete antimicrobial efficacy.

[0364] Conclusions [0365] AMC-109 can be impregnated into solid silicone products using a swell and dry method. [0366] The local antimicrobial efficacy is complete for S. aureus (ATCC 8325), S. epidermidis (RP62a), E. coli (5067-6002) and P. aeruginosa (PAO1). [0367] The results from the wash and cut experiments shows that AMC-109 is impregnated into the interior of the product, but the product still shows very good anti-colonizing and local antimicrobial efficacy.