ANTI-MICROBIAL COMPOSITIONS

Abstract

A metal-EDTA compound/complex for combatting biofilms and/or treating wounds. The compound/complex comprises EDTA and from two to four metal ions. Of those two to four metal ions, at least two are different metal ions selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ir, Mo, Rh, Ru, Ti, and Zn ions. The metal-EDTA compound/complex may exhibit any one or more of anti-microbial, anti-biofilm and anti-inflammatory activities in use and may increase the susceptibility of a biofilm and the microorganisms within said biofilm to attack by anti-microbial agents, helping to remove and sanitise the biofilm. A composition, wound dressing and medical device comprising the metal-EDTA complex are also provided. Uses of the metal-EDTA compound/complex as a medicament and/or to sanitise and/or substantially remove a biofilm from a substrate are also disclosed.

Claims

1. A compound of formula M.sub.n(EDTA); wherein: n is an integer from 2 to 4; each M is a metal ion; and Mn comprises at least two different metal ions selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ir, Mo, Rh, Ru, Ti, and Zn ions.

2. The compound according to claim 1, wherein n=3 or 4 and M.sub.n comprises two Ag ions.

3. The compound according to claim 1, wherein M.sub.n comprises at least one Ag ion and one Zn ion.

4. The compound according to claim 3, having the formula Ag.sub.2Zn(EDTA).

5. The compound according to claim 1, wherein M.sub.n comprises at least one Ag ion and one Cu ion.

6. The compound according to claim 5, having the formula Ag.sub.2Cu(EDTA).

7. A composition comprising one or more compounds according to claim 1.

8. The composition according to claim 7 which is a hydrogel which comprises the one or more compounds.

9. The composition according to claim 7, comprising fibres which are in contact with the one or more compounds.

10. The composition according to claim 7, wherein the composition comprises a non-metal ion anti-microbial agent.

11. The composition according to claim 7, wherein the composition comprises a surfactant.

12. A wound dressing comprising a compound according to claim 1.

13. A medical device comprising a compound according to claim 1.

14. (canceled)

15. A medicament comprising a compound according to claim 1.

16. A kit comprising a compound according to claim 1 and a medical device or wound dressing.

17. (canceled)

18. A wound dressing comprising a composition according to claim 7.

19. A medical device comprising a composition according to claim 7.

20. A medicament comprising a composition according to claim 7.

21. A kit comprising a composition according to claim 7 and a medical device or a wound dressing.

Description

EXAMPLES

Comparative Example 1Tetra SilverEDTA Formation and Elution

Standard Curve

Principle:

[0124] Fe.sup.3+ reacts with thiocyanate producing red complexes. Na.sub.4(EDTA) (tetra sodium EDTA) reacts with the Fe.sup.3+ resulting in a red colour that decreases as the levels of Na.sub.4EDTA increase.

Instrument:

[0125] UV spectrophotometer

Reagents:

[0126] Na.sub.4(EDTA) standard,

[0127] hydrochloric acid (HCl) ACS reagent,

[0128] thiocyanate solutionammonium thiocyanate in 100 ml water,

[0129] Fe.sup.3+ solutionformed by dissolving ammonium iron(III) sulfate dodecahydrate in a small amount of HCl, adding sodium acetate buffer and then adding to water.

TABLE-US-00001 TABLE 1 Results Optical Na.sub.4EDTA Density concentration (mM) 0.3715 0 0.33 0.85 0.3025 1.7 0.2665 2.55 0.2525 3.4 0.2165 4.25 0.1865 5.1 0.1465 6.8 0.08 8.5 0.0715 10.2 0.0355 11.9 0.0365 13.6

[0130] FIG. 1 shows the standard correlation curve for tetra sodium EDTA. The results show there was a linear correlation between OD (optical density) value and the amount of tetra sodium EDTA in the solution at concentration from 0 to 11.9 mM, which means this method can be used to measure quantitatively tetra sodium EDTA as solid or in the solution.

Tetra SilverETDA Development and Elution Measurement

Methods

[0131] 1. Prepare a polyurethane (PU) solution by dissolving 1 g PU in 10 ml dimethylformamide (DMF) at 60 C. overnight. [0132] 2. Prepare a tetra silver EDTA complex and suspend in the PU solution at different concentrations100 mg/ml, 50 mg/ml, 10 mg/ml, 1 mg/ml and 0 mg/ml. [0133] 3. Coat the tetra silver EDTA on glass coverslips and plastic discs to make films. [0134] 4. The coated coverslips and plastic discs are then immersed in 2 ml deionized water for 1 week. [0135] 5. The elution of tetra silver EDTA is determined according to the standard curve method.

Results

[0136] The PU films on glass coverslips, were named G1, G2, G3, G4, and G5 correlating to different concentrations of tetra silver EDTA complex. The elution results are shown in table 2 below.

TABLE-US-00002 TABLE 2 Tetra silver-EDTA eluted Films (g) eluted-% eluted G1 (10 mg) 3560.822-35.61 G2 (5 mg) 1755.496-35.11 G3 (1 mg) 358.2583-35.83 G4 (0.1 mg) 31.86451-31.86 G5 0

[0137] The results indicated that after one week immersed in deionised water, about 30%-36% tetra silver EDTA eluted from the films.

Comparative Example 2Ag.SUB.4.(EDTA)

[0138] Synthesis: 4Ag.sup.+(aq)+EDTA.sup.4(aq).fwdarw.Ag.sub.4(EDTA) (s)

[0139] Silver nitrate was reacted with a tetra-sodium salt of EDTA in a 4:1 molar ratio under vigorous stirring. The resulting solid was filtered off, washed with cold deionized water 3 times, washed with 50% ethanol, 70% ethanol and 100% ethanol one time each; and then dried in a vacuum oven at 50 C. in a dark flask to avoid exposure to direct light. A white powder of Ag.sub.4(EDTA) was produced in the following yield.

TABLE-US-00003 Ag.sub.4(EDTA) (s) AgNO.sub.3 Na.sub.4(EDTA) Theory Measured Weight (g) 0.680 0.380 0.720 0.605

[0140] The FTIR of EDTA, Na.sub.4(EDTA) and Ag.sub.4(EDTA) are shown in FIGS. 2A-C. The absorption band centred at 1740 cm.sup.1 in FIG. 2A (EDTA) attributed to the stretching vibration of carbonyl in COOH is not present in the spectra of FIGS. 2B and 2C (Na.sub.4EDTA and Ag.sub.4EDTA respectively). The spectrum of the pure Na.sub.4EDTA (FIG. 2B) is characterized by an absorption band centred at around 1580 cm.sup.1 which is attributed to the asymmetric stretching vibration of carbonyl in the four ionic carboxylate groups (v.sub.as COO.sup.). The corresponding symmetric stretching vibration occurs close to 1465 cm.sup.1 (v.sub.s COO.sup.). The asymmetric stretching vibration adsorption band of carbonyl in the Ag.sub.4EDTA (FIG. 2C) is much stronger than the respective peak in Na.sub.4EDTA and the band centre moves to around 1550 cm.sup.1. The corresponding symmetric stretching vibration band centred at 1465 cm.sup.1, which is a less intense absorption band than the one at 1550 cm.sup.1. The results indicated Ag.sub.4EDTA complex formed.

Ag.SUB.4.(EDTA) Suspension Coating Process

[0141] The Ag.sub.4(EDTA) powders were suspended in deionised water at 1 mg/ml. A 100 l portion of the suspension was dropped onto a first glass coverslip. A 50 l portion of the suspension was added to a 50 l portion of a 1.7% NaCl solution and dropped onto a second glass coverslip. A 50 l portion of the suspension was mixed with a 50 l portion of a PU solution and then dropped onto a third glass coverslip. All the coverslips were dried in vacuum oven at 50 C. in a dark flask to avoid exposure to direct light. The results showed that Ag.sub.4(EDTA) can be coated on glass or as films of polymers (certainly at least polyurethane).

Displacement Experiment

[0142] Adding saline to the suspension of Ag.sub.4(EDTA), the suspension quickly precipitated a solid (AgCl). The precipitate was filtered off, washed with deionized water 4 times; and then dried in a vacuum oven at 50 C. in a dark flask to avoid exposure to direct light.

TABLE-US-00004 AgCl (g) Ag.sub.4(EDTA) (g) Saline Theory Measured Weight (g) 0.1277 5 ml 0.1660 0.1784

ExamplesSilver and Copper EDTA Complex SynthesisCompounds 1 and 2

[0143] These compounds were prepared by first dissolving 38 g of ethylenediaminetetraacetic acid tetrasodium salt hydrate (Tetra sodium EDTA) in 1000 ml H.sub.2O to provide solution 1. Then 68 g of silver nitrate (AgNO.sub.3) was dissolved in 1000 ml H.sub.2O to provide solution 2. 25 g of cupric sulfate pentahydrate (CuSO.sub.4.5H.sub.2O) was dissolved in 1000 ml H.sub.2O to provide solution 3.

Preparation of Compound 1AgCuNaEDTA

[0144] 40 ml of solution 1 was mixed with 20 ml solution 2 with vigorous stirring for 10 mins. The mixture was then filtered to obtain a solid. The solid was washed with water twice. About 60 ml of solution 3 was added to the solid and the mixture stirred vigorously until the solid totally dissolved. The resultant solution was filtered with a 0.45 m syringe filter to provide the product compound 1AgCuNaEDTA.

Preparation of Compound 2Ag.SUB.2.CuEDTA

[0145] 30 ml of solution 1 was mixed with 30 ml solution 2 with vigorous stirring for 10 mins. The mixture was then filtered to obtain a solid. About 80 ml of solution 3 was added to the solid and the mixture stirred vigorously until the solid totally dissolved. The resultant solution was filtered with a 0.45 m syringe filter to provide the product compound 2Ag.sub.2CuEDTA. FIG. 3 shows the ATR-FTIR spectrum of the dark blue powder of Ag.sub.2Cu(EDTA).

ExamplesSilver and Zinc EDTA Complex SynthesisCompounds 3 and 4

[0146] These compounds were prepared by first dissolving 38 g of ethylenediaminetetraacetic acid tetrasodium salt hydrate (Tetra sodium EDTA) in 1000 ml H.sub.2O to provide solution 1. Then 68 g of silver nitrate (AgNO.sub.3) was dissolved in 1000 ml H.sub.2O to provide solution 2. 18.6 g of zinc sulfate monohydrate (ZnSO.sub.4H.sub.2O) was dissolved in 1000 ml H.sub.2O to provide solution 4.

Preparation of Compound 3AgZnNaEDTA

[0147] 40 ml of solution 1 was mixed with 20 ml solution 2 with vigorous stirring for 10 mins. The mixture was then filtered to obtain a solid. The solid was washed with water four times. About 100 ml of solution 4 was added to the solid and the mixture stirred vigorously until the solid totally dissolved. The resultant solution was filtered with a 0.45 m syringe filter to provide the product compound 3AgZnNaEDTA.

Preparation of Compound 4Ag.SUB.2.ZnEDTA

[0148] 30 ml of solution 1 was mixed with 30 ml solution 2 with vigorous stirring for 10 mins. The mixture was then filtered to obtain a solid. About 120 ml of solution 4 was added to the solid and the mixture stirred vigorously until the solid totally dissolved. The resultant solution was filtered with a 0.45 m syringe filter to provide the product compound 4Ag.sub.2ZnEDTA.

[0149] All compounds 1-4 were stored at room temperature in the dark as a precaution.

[0150] The following experiments were carried out with the following EDTA compounds/complexes prepared as described above:

[0151] Compound 1AgCuNaEDTA

[0152] Compound 2Ag.sub.2CuEDTA

[0153] Compound 3AgZnNaEDTA

[0154] Compound 4Ag.sub.2ZnEDTA

[0155] Displacement Profiles of EDTA Complexes

[0156] The displacement experiment described above was repeated for compounds 1-4 to provide the percent of the displacement of silver, copper, zinc from the EDTA complexes by physiological saline. The results are shown in Table 3 below.

TABLE-US-00005 TABLE 3 EDTA Displacement (%) compound Silver Copper Zinc 1 81.37 4.29 53.49 5.56 2 77.99 3.90 41.37 6.92 3 82.32 5.63 47.33 13.29* 4 80.77 6.45 38.86 9.01*

[0157] The results of displacement experiments demonstrated that silver, copper and zinc form compounds 1-4 can be displaced by physiological saline (sodium ions). The results (Table 3) showed that about 80% silver, 40-50% copper and zinc were displaced by the sodium in the saline.

Incorporation of EDTA Complexes into Collagen

[0158] The EDTA compounds 1-4 were added to collagen films and then investigated for elution of these complexes. The collagen film was prepared from a solution of oxidized bovine atelocollagen type I, glycerol and EDTA compounds 1-4. 1.8 ml 10% glycerol solution was added into 20 ml oxidized bovine atelocollagen type I solution. 5 ml EDTA complex were added into the solution with vigorous stirring, and the mixture was adjusted to pH=7.0 by dropwise addition of 5% NaOH. The final solution was adjusted to 30 ml by ultrapure water and was cast into PVC mould. The solution was dried in a fume hood overnight. All the collagen films, impregnated with the EDTA complexes were immersed in water, saline or simulated wound fluid.

TABLE-US-00006 TABLE 4 Elution profile of EDTA compound 4 from collagen platform in water and saline Time Elution in water (ppm) Elution in saline (ppm) (days) Silver Zinc EDTA Silver Zinc EDTA 1 19.08 0.92 9.86 0.39 9.25 0.90 20.34 1.42 5.65 0.06 9.75 1.31 2 12.61 1.04 9.60 0.33 0.96 0.64 14.49 2.63 6.26 0.67 0.47 0.35 3 10.64 1.19 8.08 0.70 1.53 1.08 15.23 2.41 6.44 0.60 0.40 0.08 4 9.56 0.79 6.24 0.40 0.24 0.28 12.93 2.10 5.58 0.33 0.13 0.90 5 10.35 0.73 6.53 0.23 1.17 1.31 13.30 2.14 5.18 0.15 0.07 0.93

[0159] The elution results of the collagen films in water and saline are shown in Table 4. For silver, the highest elution rate was on day 1 (19.080.92 ppm in water, 20.341.42 ppm in saline). Then the elution rate decreased, but the elution rate was still higher than 10 ppm in water, and 13 ppm in saline. There was more silver eluted out in saline than in water. In total 54.03% and 66.22% silver were eluted out from collagen films in water and in saline respectively over the 5 days. For zinc the highest elution rate in water was on day 1 (9.860.39 ppm). Then the elution rate slightly decreased, but kept above 6.5 ppm at day 5. However, the elution rate in saline remained constant at around 6 ppm from day 1 to day 5. Overall, 37.89% and 27.36% zinc was eluted out from collagen films in water and in saline respectively for 5 days. For EDTA, the results show that most EDTA eluted out in day 1 both in water and in saline. After day 1, only a small amount EDTA was eluted out. In total, there was 16.95% and 13.44% EDTA eluted out from collagen films in water and in saline respectively over 5 days.

Gauze Platform and Elution of EDTA Compound 4

[0160] Pieces of gauze was soaked in water, 2% EDTA, 4% EDTA and either EDTA compound 4 in high concentration (sample 1) or in low concentration (sample 2) for 1 hour; then the gauze was taken out and put into a 60 mm polystyrene culture plate and dried in fume hood for 24 hours. The gauzes were weighed before soaking, after soaking and after drying for 24 hours.

TABLE-US-00007 TABLE 5 The percentage of weight change after soaking in different solutions EDTA EDTA compound 4, compound 4, Wa- 2% 4% sample 1 sample 2 ter EDTA EDTA (EDTA-AgZn) (EDTA-AgZn) Weight 0.133 28.12 56.87 47.03 47.55 variety (%) STDEV 0.07 0.71 2.13 3.74 1.66

[0161] To assess the elution profile of the EDTA complexes from gauze, the loaded gauzes were immersed in water and saline (Table 6 and Table 7). The results showed that silver, zinc and EDTA eluted out rapidly at day 1 and day 2 in both water and saline. After day 2, the eluting of silver, zinc and EDTA decreased more than 5 times compared to day 1.

TABLE-US-00008 TABLE 6 Elution profile of EDTA compound 4, sample 1 from loaded gauze in water and saline Time Elution in water (ppm) Elution in saline (ppm) (days) Silver Zinc EDTA Silver Zinc EDTA 1 35.89 5.66 21.99 2.85 18.09 3.19 49.91 2.29 29.69 2.40 24.65 2.40 2 25.27 5.63 15.66 2.73 12.54 3.11 12.76 4.44 9.14 2.77 9.19 3.42 3 4.93 2.56 4.42 1.62 1.90 0.79 3.43 0.54 3.71 0.27 1.83 0.26 4 1.75 0.76 3.26 0.35 1.27 0.84 2.29 1.73 3.42 0.93 1.28 0.84 5 0.85 0.34 2.06 1.21 0.87 0.36 2.52 2.26 2.68 2.00 0.45 0.15

TABLE-US-00009 TABLE 7 Elution profile of EDTA compound 4, sample 2 from loaded gauze in water and saline Time Elution in water (ppm) Elution in saline (ppm) (days) Silver Zinc EDTA Silver Zinc EDTA 1 23.32 3.95 17.08 3.09 13.23 1.28 27.32 3.84 22.71 1.75 17.31 1.77 2 16.28 3.75 12.78 1.55 8.74 2.01 8.36 2.37 7.67 2.03 6.84 2.67 3 3.82 1.39 4.21 1.19 1.55 0.56 2.55 0.94 3.70 0.20 1.75 0.31 4 1.42 0.40 3.37 0.26 1.13 0.57 1.46 0.99 3.48 0.68 1.11 0.61 5 0.59 0.35 2.49 0.88 0.79 0.19 1.57 1.43 2.95 1.46 0.54 0.11
Incorporation of EDTA Complexes into Hydrogels

[0162] Hydrogels were loaded with EDTA compounds and investigated for elution of the silver, zinc and EDTA. Carbopol ETD 2020 was obtained from Lubrizol Advanced Materials, Inc. USA. 1.2 g carbopol power was dissolved in 42.36 g deionized water for 1 hour to fully hydrate. Then 8.4 ml of the EDTA complexes and 4.8 g glycerine were added into carbopol solution with vigorous stirring; finally, 3 g 50% triethanolamine was dropped into the suspension to obtain final products: carbopol hydrogel with EDTA complexes.

[0163] The cumulative elution profiles of the EDTA complexes from the hydrogels into water and saline are shown in Table 8 and Table 9. The results show that silver, zinc and EDTA eluted out rapidly in day 1 and there was no distinct cumulative eluting after day 1 in water. The silver, zinc and EDTA increased in saline day by day, which indicated the release of silver, zinc and EDTA can be controlled from the hydrogel.

TABLE-US-00010 TABLE 8 Cumulative Elution profile of EDTA compound 4, sample 1 loaded onto hydrogel into water and saline Time Elution in water (ppm) Elution in saline (ppm) (days) Silver Zinc EDTA Silver Zinc EDTA 1 44.39 5.98 31.88 3.53 31.12 5.40 32.09 3.02 22.12 3.31 26.39 2.06 2 47.13 5.03 36.09 3.61 26.21 4.20 37.09 2.91 30.90 5.81 27.18 2.34 3 45.58 4.54 34.28 4.55 37.28 2.58 47.05 2.79 36.73 3.64 35.02 0.72 4 46.88 6.86 33.78 2.20 37.53 3.27 51.24 1.53 40.31 1.84 37.71 1.92 5 45.91 7.04 35.99 1.16 35.11 5.39 59.55 1.21 43.22 3.91 40.57 1.41

TABLE-US-00011 TABLE 9 Cumulative Elution profile of EDTA compound 4, sample 2 loaded onto hydrogel in water and saline Time Elution in water (ppm) Elution in saline (ppm) (days) Silver Zinc EDTA Silver Zinc EDTA 1 28.41 3.48 20.77 3.25 20.80 3.07 21.10 1.80 15.30 2.12 16.64 1.41 2 29.03 4.71 23.57 2.12 22.50 2.86 26.06 2.03 20.51 3.94 17.31 1.43 3 29.12 2.73 22.53 7.30 24.64 1.57 31.27 0.96 23.98 1.92 21.81 0.42 4 29.89 3.51 22.23 1.97 24.30 1.51 32.50 0.96 26.11 1.08 22.70 1.70 5 30.64 5.25 23.54 1.23 23.03 3.46 37.54 0.61 27.28 3.01 25.02 0.96

Silver Content

[0164] 1 g hydrogel was placed in 10 ml 37% nitric acid to dissolve. After filtration, the solution was diluted to an exact volume of 0.5 ml. The diluted solutions were mixed with 0.5 ml 1% sodium dodecyl sulphate (SDS) and 0.5 ml of 1 M sulphuric acid followed by the addition of 0.5 ml 110.sup.3 M dithizone solution. The absorbance was measured at 490 nm against a corresponding reagent blank. The silver content in an unknown sample was determined using a concurrently prepared calibration graph. The concentration of silver was then measured against series of calibration standards by modified spectrophotometry. Concentration is then converted to a weight % based on the weight of dressing used.

[0165] The results of the silver content experiments on the hydrogel measured by spectrophotometry are shown in the Table 10. The results show that the low level silver hydrogel had 0.095% silver only, there was 0.101% silver (w/w) in the EDTA compound 4, sample 1 (high levels of EDTA-AgZn) hydrogel and 0.046% silver (w/w) in the other EDTA compound 4, sample 1 hydrogel (low levels of EDTA-AgZn).

TABLE-US-00012 TABLE 10 The silver content in the hydrogels (%) Control EDTA compound 4, EDTA compound 4, Hydrogel Silver Hydrogel sample 2 (low) sample 1 (high) 0 0.095 0.006 0.046 0.008 0.101 0.021

Water Donation/Absorption

[0166] Table 11 shows that all the hydrogels belong to 1 b or 1c type. That means all the hydrogels are donation type. The tetra sodium EDTA only hydrogel was found to be in the 1c hydrogel group (donated the most fluid); other gels were characterised as 1 b gels, which donated 5 to 10% water from hydrogel to gelatin.

TABLE-US-00013 TABLE 11 Water donation/absorption results Mean % decrease Mean % in hydrogel increase in weight hydrogel weight (Gelatin Gel Hydrogel (Agar as test gel) as test gel) type Control Hydrogel 3.17 0.83 9.85 0.44 1b Silver Hydrogel (0.1%) 2.10 0.14 8.81 0.56 1b Zinc Hydrogel (0.06%) 0.18 0.13 7.97 1.33 1b Tetrasodium EDTA Hydrogel 2.99 1.22 10.16 2.71 1c (0.2%) EDTA compound 4, sample 1.56 0.29 5.33 0.94 1b 2 Hydrogel (EDTA-ZnAg - low - 0.05% silver) EDTA compound 4, sample 2.43 0.47 5.29 0.40 1b 1 Hydrogel (EDTA-ZnAg - high - 0.1% silver)

Microbiological Analysis of the EDTA Complexes

Microbiological Evaluation of Tetra Sodium EDTA

[0167] Initial testing on tetrasodium EDTA showed tetrasodium EDTA was ineffective as a treatment against immature and mature S. aureus and P. aeruginosa biofilms at short contract times (Table 12, Table 13 and Table 14). The incorporation of tetrasodium EDTA into fibrous dressing frameworks did however result in a 3-Log reduction, respectively, in S. aureus and P. aeruginosa biofilm following 24 hours of treatment (Table 15 and Table 15).

TABLE-US-00014 TABLE 12 Efficacy of tetrasodium EDTA against P. aeruginosa and S. aureus 24-hour biofilm MIC MBC MBEC 1 min 30 min 1 h 24 h 1 min 30 min 1 h 24 h 1 min 30 min 1 h 24 h P. aeruginosa 25 12.5 25 6.25 50 50 25 6.25 x x 6.25 6.25 S. aureus 25 12.5 25 6.25 50 50 25 6.25 x x 6.25 6.25

[0168] Minimum inhibitory concentration (MIC), minimum biocidal concentration (MBC) and minimum biofilm eradication (MBEC) were determined. Values represent a percentage of the stock antimicrobial solution (8%). The x denotes that all concentrations of the antimicrobial tested, including the antimicrobial in neat form, were ineffective.

TABLE-US-00015 TABLE 13 Log reduction (LR) and percentage kill (%) values of S. aureus biofilms when exposed to tetra sodium EDTA S. aureus 24-Hour Biofilms 72-Hour Biofilms Treatment Time 1 5 15 1 5 15 Antimicrobial Kill Kill Kill Kill Kill Kill LR (%) LR (%) LR (%) LR (%) LR (%) LR (%) Tetrasodium 1.59 1.56 97.41 0.40 0.05 59.90 0.71 0.11 80.51 0.34 0.37 54.09 0.70 0.05 79.86 0.70 0.02 80.25 EDTA (4%) Untreated Mean Log.sub.10 density of 6.93 0.05 Mean Log.sub.10 density of 7.58 0.05 control LR - 0 LR - 0 Kill (%) - 0 Kill (%) - 0

[0169] Mean Log values were calculated and the standard error (SE) of the mean LR was determined (LRSE). The Mean Log.sub.10 density of untreated controls was calculated (standard deviation).

TABLE-US-00016 TABLE 14 Log reduction (LR) and percentage kill (%) values of P. aeruginosa biofilms when exposed to tetra sodium EDTA at varying concentrations. P. aeruginosa 24-Hour Biofilms 72-Hour Biofilms Treatment Time 1 5 15 1 5 15 Antimicrobial Kill Kill Kill Kill Kill Kill LR (%) LR (%) LR (%) LR (%) LR (%) LR (%) Tetrasodium 0.77 0.08 83.018 1.68 0.05 97.92 2.31 0.10 99.51 2.10 0.05 99.22 2.35 0.02 99.56 1.31 0.12 95.07 EDTA (4%) Untreated Mean Log.sub.10 density of 7.62 0.03 Mean Log.sub.10 density of 7.83 0.15 control LR - 0 LR - 0 Kill (%) - 0 Kill (%) - 0

[0170] Mean Log values were calculated and the standard error (SE) of the mean LR was determined (LRSE). The Mean Log.sub.10 density of untreated controls was calculated (standard deviation).

TABLE-US-00017 TABLE 15 The antimicrobial activity of tetra sodium EDTA (at varying concentrations) - incorporated fibrous dressing against S. aureus using the direct contact method Time 24 Time 24 Log.sub.10 Log.sub.10 Microbicidal/ Dressing Density SD Reduction Microbistatic Untreated fibrous dressing 8.88 0.05 0 Microbistatic (Negative control) Durafiber 8.85 0.15 0.90 Microbistatic AQUACEL Ag (Positive 2.88 0 6.88 Microbicidal control) Fibrous Dressing 1 6.35 0.26 3.40 Microbicidal Fibrous Dressing 2 6.28 0.05 3.47 Microbicidal Fibrous Dressing 3 6.31 0.03 3.44 Microbicidal Fibrous Dressing 4 8.74 0.07 1.01 Microbistatic Fibrous Dressing 5 8.80 0.07 0.95 Microbistatic Fibrous Dressing 6 9.02 0.02 0.73 Microbistatic

[0171] Results for the antimicrobial effectiveness of the dressing were expressed as microbicidal (Log.sub.10 Reduction 3) or microbistatic (Log.sub.10 Reduction 3). SD represents standard deviation. Experiment was performed on triplicate dressings.

TABLE-US-00018 TABLE 16 The antimicrobial activity of EDTA-incorporated fibrous dressing against P. aeruginosa using the direct contact method Time 24 Time 24 Log.sub.10 Log.sub.10 Microbicidal/ Dressing Density SD Reduction Microbistatic Untreated chitosan 9.75 0.59 0 Microbistatic (Negative control) Durafiber 9.57 0.53 0.18 Microbistatic AQUACEL Ag 2.30 0.35 7.45 Microbicidal (Positive control) Fibrous Dressing 1 6.41 0.95 3.34 Microbicidal Fibrous Dressing 2 6.45 1.01 3.31 Microbicidal Fibrous Dressing 3 9.21 0.07 0.54 Microbistatic Fibrous Dressing 4 9.46 0.11 0.29 Microbistatic Fibrous Dressing 5 9.80 0.03 0.05 Microbistatic Fibrous Dressing 6 9.63 0.07 0.12 Microbistatic

[0172] Results for the antimicrobial effectiveness of the dressing were expressed as microbicidal (Log.sub.10 Reduction 3) or microbistatic (Log.sub.10 Reduction 3). SD represents standard deviation. Experiment was performed on triplicate dressings.

Microbiological Evaluation of EDTA Complexes (EDTA-AgZn and EDTA-AgCu)

[0173] Different EDTA complexes achieved complete kill in S. aureus and P. aeruginosa biofilms (Table 17).

TABLE-US-00019 TABLE 17 Number of colonies detected following exposure of 24-hour CDC biofilms to different EDTA compounds: EDTA-Cu, EDTA-Ag.sub.2Cu (compound 2), EDTA-Ag.sub.2Zn (compound 4), EDTA-AgZnNa (compound 3). Compound CuNa- Ag.sub.2Cu- Ag.sub.2Zn- AgZnNa- EDTA EDTA EDTA EDTA Microorganism Cfu/ml Cfu/ml Cfu/ml Cfu/ml S. aureus 0 0 0 0 P. aeruginosa 0 0 0 0

[0174] EDTA compounds 1-4 (prepared as described above) were tested against P. aeruginosa and S. aureus biofilms at a concentration of 100 ppm silver, to which the compounds Ag/Cu/NaEDTA and Ag/Zn/NaEDTA showed 100% kill in S. aureus and at a 3.5 log reduction in P. aeruginosa (Table 18).

TABLE-US-00020 TABLE 18 Log reduction (LR) and percentage kill (%) values of S. aureus and P. aeruginosa. P. aeruginosa S. aureus EDTA compound LR Kill (%) LR Kill (%) 1 3.88 0.15 99.99 N.D 100 3 3.51 0.04 99.97 N.D 100 2 3.31 0.01 99.95 4.0 0.20 99.99 4 3.34 0.11 99.95 3.73 0.18 99.98 Untreated control 0 0.07 0 0 0.01 0

[0175] Microbial cultures were exposed to compounds for 24 hours. Compounds were diluted according to 100 ppm of silver. Biofilms were formed using the CDC bioreactor. Samples were performed in triplicate. Mean Log values were calculated and the standard error (SE) of the mean LR was determined (LRSE). Given that the log of zero is mathematically undefined, in cases whereby no colonies were detected following antimicrobial treatment, N.D for not determined is used.

[0176] The anti-biofilm efficacy of the EDTA-AgZn compound as a liquid was evaluated using the MBEC model. 100% kill of P. aeruginosa biofilms (no viable colonies detected) following treatment with various concentrations of the complexes (1000 ppm-62.5 ppm) was observed (Table 19).

TABLE-US-00021 TABLE 19 Log reduction values related to the treatment of S. aureus and P. aeruginosa 48-hour biofilms with EDTA-Ag.sub.2Zn compound (EDTA compound 4) Treatment time 1 hours 24 hours Treatment LR % Kill LR % Kill 1000 ppm N.D 100 N.D 100 500 ppm N.D 100 N.D 100 250 ppm N.D 100 N.D 100 125 ppm N.D 100 N.D 100 62.5 ppm N.D 100 N.D 100 P. aeruginosa control 6.19 0.05 6.85 0.15 (Mean Log density)

[0177] Biofilms were formed using the MBEC plate method. Log reduction (LR) values are expressed with standard error. N.D (not determined) is stated when no colonies were detected upon enumeration.

Anti-Biofilm Ability (MBEC)

[0178] The MBEC model (ASTM E2799) was used to assess the anti-biofilm efficacy of the liquid formulas. Biofilms were grown on polystyrene pegs in the MBEC model for either 24 or 48 hours before treatment. Using the liquid form of the EDTA complexes 24- and 48-hour biofilms were tested (Table 20). The results show that the complexes and silver solution (50 ppm and 100 ppm) caused 100% kill of 24- and 48-hour biofilms. Similarly, 4% tetrasodium EDTA caused 100% kill in 24 and 48 hours on S. aureus, MRSA, S. epidermidis and E. faecalis biofilms. However for P. aeruginosa 24- and 48-hour biofilms there was a 5.943.43 and 6.994.04 log reduction respectively. The increase in log reduction for 48-hour biofilms when compared with 24-hour biofilms highlights the efficacy of the EDTA complexes as an anti-biofilm agent.

TABLE-US-00022 TABLE 20 MBEC results for tetrasodium EDTA and EDTA compound 4. 24-Hour Biofilm 48-Hour Biofilm Treatment LR Kill % LR Kill % S. aureus ATCC 29213 Control 0 0 0 0 0 0 tEDTA (4%) 6.44 100 6.50 100 Silver 100 ppm 6.44 100 6.50 100 Silver 50 ppm 6.44 100 6.50 100 EDTA-Ag.sub.2Zn (compound 4) 100 ppm 6.44 100 6.50 100 EDTA-Ag.sub.2Zn (compound 4) 50 ppm 6.44 100 6.50 100 S. aureus MRSA BAA-43 control 0 0 0 0 0 0 tEDTA (4%) 5.40 100 6.43 100 Silver 100 ppm 5.40 100 6.43 100 Silver 50 ppm 5.40 100 6.43 100 EDTA-Ag.sub.2Zn (compound 4) 100 ppm 5.40 100 6.43 100 EDTA-Ag.sub.2Zn (compound 4) 50 ppm 5.40 100 6.43 100 P. aeruginosa NCTC 10662 control 0 0 0 0 0.03 0 tEDTA (4%) 5.94 3.43 99.9998 6.99 4.04 99.9999 Silver 100 ppm 7.44 100 8.44 100 Silver 50 ppm 7.44 100 8.44 100 EDTA-Ag.sub.2Zn (compound 4) 100 ppm 7.44 100 8.44 100 EDTA-Ag.sub.2Zn (compound 4) 50 ppm 7.44 100 8.44 100 S. epidermidis ATCC 35984 0 0 0 0 0 0 tEDTA (4%) 6.09 100 6.41 100 Silver 100 ppm 6.09 100 6.41 100 Silver 50 ppm 6.09 100 6.41 100 EDTA-Ag.sub.2Zn (compound 4) 100 ppm 6.09 100 6.41 100 EDTA-Ag.sub.2Zn (compound 4) 50 ppm 6.09 100 6.41 100 E. faecalis ATCC 29212 0 0 0 0 0 0 tEDTA (4%) 6.35 100 6.40 100 Silver 100 ppm 6.35 100 6.40 100 Silver 50 ppm 6.35 100 6.40 100 EDTA-Ag.sub.2Zn (compound 4) 100 ppm 6.35 100 6.40 100 EDTA-Ag.sub.2Zn (compound 4) 50 ppm 6.35 100 6.40 100

[0179] 24- and 48-hour biofilms were formed using MBEC plate method and treated for 24 hours. Technical replicates n=3. Log reduction (LR) values showstandard error (SE) of LR value. The Log densitiesstandard deviation for untreated controls (24-hour and 48-hour biofilms) are: P. aeruginosa NCTC 10662 (7.440.06 and 8.440.034), S. aureus ATCC 29213 (6.450.05 and 6.500.38), S. aureus MRSA BAA-43 (5.400.41 and 6.430.25), E. faecalis ATCC 29212 (6.350.04 and 6.400.09) and S. epidermidis ATCC 35984 (6.090.36 and 6.410.62).

Impregnation of Gauze with the Complexes

[0180] Gauze impregnated with the EDTA-Ag.sub.2Zn compound 4 showed complete biofilm kill after 24 hours (see Table 21), which correlated with elution data whereby the largest amount of silver and EDTA eluted at 24 hours. Furthermore, gauze containing the EDTA-Ag.sub.2Zn compound 4 was more effective than tetrasodium EDTA at 2% and 4%. There was a greater log reduction in biofilms when treated with gauze containing 4% EDTA than 2% EDTA. The collagen film containing the compound was less effective than the gauze.

TABLE-US-00023 TABLE 21 Log reduction (LR) and percentage kill (%) values of S. aureus and P. aeruginosa biofilms following treatment in the filter model. P. aeruginosa S. aureus Treatment LR Kill (%) LR Kill (%) Control gauze 1.84 1.06 98.55 0.42 0.24 61.75 Gauze 2% tEDTA 2.05 1.18 99.11 0.56 0.32 72.43 Gauze 4% tEDTA 3.40 1.96 99.96 1.22 0.70 93.98 Gauze + N.D 100 N.D 100 EDTA-AgZn compound Collagen film + 2.85 1.64 99.86 4.04 2.33 99.991 EDTA-Ag.sub.2Zn (compound 4) Untreated control Mean log density: 10.20 Mean log density: 9.88

[0181] Biofilms (48-hours) were exposed to various platforms for 24 hours. The compound used to impregnate the platforms was EDTA-Ag.sub.2Zn (compound 4). Samples were run in triplicate. Mean Log values were calculated and the standard error (SE) of the mean LR was determined (LRSE). In cases whereby no colonies were detected following treatment, N.D for not determined is used, which represents 100% kill. Table 21 provides evidence that the complexes of the present invention demonstrate potential synergy between agents and out performed tetra sodium EDTA.

Addition of the Complexes to a Hydrogel

[0182] The CDC biofilm bioreactor model (ASTM E2871) was used to test anti-biofilm efficacy of the hydrogel formulations. Biofilms were grown on polycarbonate coupons in the bioreactor for 48 hours (48-hour) biofilm. The formulation of a hydrogel containing the complexes showed complete biofilm kill in the CDC model after 24 hours of treatment (see Table 22). The hydrogel containing the compound 4 outperformed the hydrogel containing silver alone.

TABLE-US-00024 TABLE 22 Log reduction (LR) and percentage kill (%) values of S. aureus and P. aeruginosa biofilms following treatment in the CDC model. P. aeruginosa S. aureus Treatment LR Kill (%) LR Kill (%) Control hydrogel 1.38 0.80 95.81 0 0 Hydrogel + silver 5.13 2.96 99.9992 0 0 Hydrogel + compound 4 N.D 100 ND 100 Untreated control Mean log density: 8.97 Mean log density: 4.70

[0183] Biofilms (48-hours) were exposed to hydrogel for 24 hours. The compound used to impregnate the platforms was EDTA-Ag.sub.2Zn (compound 4). Samples were run in triplicate. Mean Log values were calculated and the standard error (SE) of the mean LR was determined (LRSE). In cases whereby no colonies were detected following treatment, N.D for not determined is used, which represents 100% kill. Table 22 demonstrates evidence that the complex of EDTA-Ag.sub.2Zn outperformed a silver hydrogel alone in antibiofilm activity.

[0184] To test the anti-biofilm efficacy of the hydrogel EDTA-Ag.sub.2Zn compound 4, we tested the hydrogel against five microorganisms in biofilm form using the CDC biofilm bioreactor. The hydrogel control showed a slight log reduction in microbial numbers. Hydrogel containing silver resulted in 100% kill in S. aureus, MRSA, S. epidermidis and E. faecalis. However the silver hydrogel only caused a 3.161.83 log reduction in P. aeruginosa (Table 23). The hydrogel containing zinc caused between a 1.24 to 1.84 log reduction respectively in all microorganisms except for S. epidermidis, whereby 100% kill was recorded. Similarly, hydrogel containing tetrasodium EDTA caused a slight log reduction in most microorganisms, however caused a 100% kill in S. epidermidis. The hydrogel formulation containing a low concentration of the compound 4 (EDTA-Ag.sub.2Zn) did not have a significant reduction in most microorganisms but did cause a 100% kill in S. epidermidis. The high concentration of the compound 4 incorporated into the hydrogel caused 100% kill in all microbial biofilms tested.

TABLE-US-00025 TABLE 23 Log reduction (LR) and percentage kill (%) values of 48-hour biofilms following treatment with hydrogel formulations in the CDC model. Biofilms (48-hours) were exposed to hydrogel for 24 hours at 37 C. 2 C. The compound used to impregnate the platforms was EDTA-Ag.sub.2Zn (compound 4) at a low and high concentration. Samples were run in triplicate. Mean Log values were calculated and the standard error (SE) of the mean LR was determined (LR SE). The mean log density (mean log density standard deviation) of each microorganism is stated. P. aeruginosa S. aureus MRSA S. epidermidis E. faecalis Kill Kill Kill Kill Kill Treatment LR (%) LR (%) LR (%) LR (%) LR (%) Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 control Control 1.15 0.66 92.89 0.79 0.45 83.62 0.82 0.47 84.92 0.20 0.12 37.40 0.44 0.25 63.67 hydrogel Hydrogel + 3.16 1.83 99.93 5.47 100 5.71 100 5.54 100 5.99 100 silver (0.1%) Hydrogel + 1.49 0.84 96.53 1.84 1.06 98.56 1.80 1.04 98.43 5.54 100 1.24 0.72 94.24 zinc (0.06%) Hydrogel + 1.66 0.96 97.80 1.64 0.95 97.71 1.38 0.79 95.78 5.54 100 1.68 0.97 97.90 tEDTA (0.2%) Hydrogel + 1.58 0.91 97.36 1.46 0.85 96.56 1.50 0.86 96.81 5.54 100 1.52 0.88 97.00 compound 4 EDTA- AgZn (low - 0.05% silver) Hydrogel + 7.53 100 5.47 100 5.71 100 5.54 100 5.99 100 complex - EDTA-AgZn (high - 0.1% silver) Hydrogel Mean log Mean log Mean log Mean log Mean log control density: 7.53 0.06 density: 5.47 0.05 density: 5.71 0.05 density: 5.54 0.11 density: 5.99 0.17

[0185] Table 23 demonstrates that the compound 4 (EDTA-Ag.sub.2Zn) outperformed a silver hydrogel alone, a zinc hydrogel alone and to tetra sodium EDTA alone, in antibiofilm activity.

Cellular Data

[0186] Direct cytotoxicity was tested using the Neutral Red Uptake assay, whereby uptake of Neutral Red by cells is proportional to the cell viability (Borenfreund, E. & Puerner, J. A. 1985, A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90), Methods in Cell Science, 9, 7-9). According to the international standard ISO 10993-5 (Wallin, R. F. & Arscott, E. 1998, A practical guide to ISO 10993-5: Cytotoxicity. Medical Device and Diagnostic Industry, 20, 96-98), if the percentage viability is less than 70% of the untreated control, then the substance is considered cytotoxic. Results showed that increasing concentrations of tetrasodium EDTA were cytotoxic to cells with percentage viability between 3.87% and 14.23%. All concentrations of Compounds 1 and 3 were classed as cytotoxic, with viability ranging from 52.57% to 53.66% and 46.46% to 63.08%, respectively. Compound 2 at all concentrations was interpreted as non-cytotoxic with percentage viability ranging from 72.01% and 72.21%. Compound 4 at 50 ppm was also non-cytotoxic, will 72.93% viability, however other concentrations were cytotoxic (Table 24). The agar diffusion method for indirect cytotoxicity allows for the qualitative assessment of cytotoxicity. According to ISO 10993-5, an achievement of a numerical grade greater than 2 is considered a cytotoxic effect.

TABLE-US-00026 TABLE 24 Percentage cell viability using the direct contact method. Direct cytotoxicity was performed using Neutral Red on EDTA compounds 1-4. Viability was expressed as the percentage of control standard deviation. Samples were tested in triplicate. Percentage of Control/ Sample Percentage Viability (%) Interpretation DMEM (Negative control) 100 0 Non cytotoxic SDS 0.2 mg/ml 6.57 0 Cytotoxic SDS 0.15 mg/ml 6.57 0 Cytotoxic SDS 0.1 mg/ml 6.57 0 Cytotoxic SDS 0.05 mg/ml 6.57 0 Cytotoxic EDTA 2% 3.87 0.66 Cytotoxic EDTA 4% 4.79 1.74 Cytotoxic EDTA 8% 7.03 1.21 Cytotoxic EDTA 10% 10.22 7.23 Cytotoxic EDTA 15% 14.23 13.02 Cytotoxic Compound 1 100 ppm 52.57 8.97 Cytotoxic Compound 1 50 ppm 53.66 9.48 Cytotoxic Compound 1 25 ppm 51.54 9.44 Cytotoxic Compound 3 100 ppm 46.46 2.40 Cytotoxic Compound 3 50 ppm 63.08 8.47 Cytotoxic Compound 3 25 ppm 50.20 17.08 Cytotoxic Compound 2 100 ppm 72.09 17.57 Non cytotoxic Compound 2 50 ppm 72.01 7.41 Non cytotoxic Compound 2 25 ppm 72.21 3.74 Non cytotoxic Compound 4 100 ppm 56.41 1.83 Cytotoxic Compound 4 50 ppm 72.93 12.90 Non cytotoxic Compound 4 25 ppm 62.67 20.39 Cytotoxic

[0187] Table 24 demonstrates that the compounds of the present invention were significantly lower in toxicity when compared to tetra sodium EDTA alone.

[0188] The assessment of the Zone Index for EDTA at all concentrations showed cytotoxicity, with zones of no neutral red uptake extended 0.5 cm to 1 cm beyond the filter disc. Despite this finding, the Lysis Index interpretation showed that EDTA at 2%, 4% and 8% was not cytotoxic to cells, whereas 10% and 15% concentrations were cytotoxic. Compound 1 at 100 ppm and 50 ppm was interpreted as cytotoxic to cells according to the zone index, whereby the zone of cells showing no neutral red uptake extended 0.5 cm to 1 cm beyond the filter disc specimen. However these concentrations were not deemed cytotoxic when interpreting the Lysis Index. Compound 1 at 25 ppm was not cytotoxic to cells. Compound 3 at 100 ppm resulted in cell cytotoxicity according to the Zone Index but not the Lysis Index. Compounds 2 and 4 at all concentrations were not cytotoxic to cells (Table 25).

TABLE-US-00027 TABLE 25 Zone index and lysis index for indirect cytotoxicity test of EDTA compounds 1-4. Zone index measures the clear zone in which cells do not stain with neutral red. The lysis index measures the number of cells affected within the zone of toxicity. All samples were tested in triplicate. Zone Lysis Sample Index Interpretation Index Interpretation DMEM 0 Non cytotoxic 0 Non cytotoxic Water 0 Non cytotoxic 0 Non cytotoxic Phenol 5 Cytotoxic 3 Cytotoxic EDTA 2% 3 Cytotoxic 2 Non cytotoxic EDTA 4% 3 Cytotoxic 1 Non cytotoxic EDTA8% 3 Cytotoxic 2 Non cytotoxic EDTA 10% 3 Cytotoxic 3 Cytotoxic EDTA 15% 3 Cytotoxic 3 Cytotoxic Compound 1 100 ppm 3 Cytotoxic 1 Non cytotoxic Compound 1 50 ppm 3 Cytotoxic 1 Non cytotoxic Compound 1 25 ppm 2 Non cytotoxic 1 Non cytotoxic Compound 3 100 ppm 3 Cytotoxic 1 Non cytotoxic Compound 3 50 ppm 2 Non cytotoxic 1 Non cytotoxic Compound 3 25 ppm 2 Non cytotoxic 1 Non cytotoxic Compound 2 100 ppm 2 Non cytotoxic 1 Non cytotoxic Compound 2 50 ppm 2 Non cytotoxic 1 Non cytotoxic Compound 2 25 ppm 2 Non cytotoxic 1 Non cytotoxic Compound 4 100 ppm 2 Non cytotoxic 1 Non cytotoxic Compound 4 50 ppm 2 Non cytotoxic 1 Non cytotoxic Compound 4 25 ppm 2 Non cytotoxic 1 Non cytotoxic

[0189] Table 25 demonstrates that the complexes were significantly lower in toxicity when compared to tetra sodium EDTA alone.

[0190] The efficacy of the EDTA-Ag.sub.2Zn compound (compound 4) in a wound biofilm model. At day 6, the untreated S. aureus wound biofilm was present in more than 50% of the tissue. After treatment with 100 ppm of the AgZn.sub.2-EDTA (compound 4), there appeared to be a reduced amount of microorganisms within the tissues, however there was no identifiable epidermis and the nuclei of fibroblasts in the dermal layer were faint.

[0191] Enzyme-linked immunosorbent assay (ELISA) was used to assess the secreted levels of the inflammatory cytokine interleukin-6 (IL-6) according to previously published methods (Foster, A. M., Baliwag, J., Chen, C. S., Guzman, A. M., Stoll, S. W., Gudjonsson, J. E., Ward, N. L. & Johnston, A. 2014, IL-36 promotes myeloid cell infiltration, activation, and inflammatory activity in skin, The Journal of Immunology, 192, 6053-6061). ELISA detection of secreted IL-6 in the same experiment showed a reduction in IL-6 in the S. aureus wound biofilm control (FIG. 4). Treatment of wound biofilm with the AgZn.sub.2-EDTA (compound 4), was shown to reduce the presence of microorganisms upon histological examination. The IL-6 levels were reduced further when compared to the S. aureus wound biofilm control.

[0192] FIG. 4 shows IL-6 cytokine secretion from wounded, biofilm-containing Labskin following treatment with liquid AgZn.sub.2-EDTA (compound 4). ELISA was used to assess the levels of IL-6 in conditioned cell culture medium. Culture medium was changed at day 4, which accounts for the reduction in IL-6. Biofilms were treated with either 50 ppm or 100 ppm of EDTA compound 4. Tests were performed in duplicate. Error bars indicate standard deviation.

[0193] FIG. 4 demonstrates that the AgZn.sub.2-EDTA (compound 4) at 100 ppm caused a significant reduction in the inflammatory marker.

[0194] In summary, the present invention provides a metal-EDTA compound/complex for combatting biofilms and/or treating wounds. The compound/complex comprises EDTA and from two to four metal ions. Of those two to four metal ions, at least two are different metal ions selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ir, Mo, Rh, Ru, Ti, and Zn ions. The metal-EDTA compound/complex may exhibit any one or more of anti-microbial, anti-biofilm and anti-inflammatory activities in use and may increase the susceptibility of a biofilm and the microorganisms within said biofilm to attack by anti-microbial agents, helping to remove and sanitise the biofilm. A composition, wound dressing and medical device comprising the metal-EDTA complex are also provided. Uses of the metal-EDTA compound/complex as a medicament and/or to sanitise and/or substantially remove a biofilm from a substrate are also disclosed.

[0195] Although a few preferred embodiments have been shown and described, it will be appreciated by those skilled in the art that various changes and modifications might be made without departing from the scope of the invention, as defined in the appended claims.

[0196] Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0197] All of the features disclosed in this specification (including any accompanying claims, and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[0198] Each feature disclosed in this specification (including any accompanying claims, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0199] The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.