COMPOSITION FOR A WOUND DRESSING

Abstract

The present invention relates to a composition that can be used as or as part of a wound dressing and to wound dressings comprising the same. More specifically, the present invention relates to a composition that disrupts and kills bacteria within a biofilm and also prevents biofilm formation. The solid composition comprises a first component selected from the group consisting of chitosan, chitin, derivatives of chitosan, derivatives of chitin, and combinations thereof; and at least one triprotic acid.

Claims

1-27. (canceled)

28. A solid composition comprising a first component selected from the group consisting of chitosan, chitin, derivatives of chitosan, derivatives of chitin, and any combination thereof; and at least one triprotic acid.

29. A composition as claimed in claim 28, wherein the first component is at least partially coated with the triprotic acid.

30. A composition as claimed in claim 28, wherein the triprotic acid is present in an amount of at least 10% of the first component.

31. A composition as claimed in claim 28, wherein the composition is insoluble in physiological fluid.

32. A composition as claimed in claim 28, wherein the first component is non-antimicrobial.

33. A composition as claimed in claim 28, wherein the ratio of the first component to the at least one triprotic acid is at least 2:1.

34. A composition as claimed in claim 28, wherein the first component is chitosan.

35. A composition as claimed in claim 34, wherein the chitosan has a degree of de-acetylation of at least 70%.

36. A composition as claimed in claim 34, wherein the first component has a viscosity greater than 150 cps in 1% acetic acid solution.

37. A composition as claimed claim 28, wherein the triprotic acid is citric acid.

38. A composition as claimed claim 28, wherein the first component is in the form of fibres, granules, flakes, powder, or any combination thereof

39. A composition as claimed in claim 28, wherein the triprotic acid is present in an amount of around 2 to 75% of the first component.

40. A composition as claimed in claim 28, wherein the triprotic acid is present in an amount of around 25 to 60% of the first component.

41. A composition as claimed in claim 28, wherein the composition is in the form of granules, flakes, fibres, powder, nonwoven textile or knitted textile.

42. A composition as claimed in claim 28, wherein the triprotic acid is coated onto a carrier material.

43. A composition as claimed in claim 28, further comprising an additional component selected from the group consisting of antimicrobial agents; pharmaceutical agents; chelating agents; wetting agents; growth factors; cytokines; agents which absorb agents which delay healing such as MMP's (matrix metalloproteinases) and elastase; calcium; vitamin K; fibrinogen; thrombin; factor VII; factor VIII; clays; oxidised regenerated cellulose; gelatin; and collagen.

44. A wound dressing comprising a composition as claimed in claim 28.

45. A composition as claimed in claim 28 for use as a therapeutic agent.

46. A composition as claimed in claim 28 for use in the treatment of wounds; or for use in disrupting and killing bacteria in a biofilm; or for use in preventing the formation of a biofilm.

47. A method of manufacturing a composition comprising a first component selected from the group consisting of chitosan, chitin, derivatives of chitosan, derivatives of chitin, and combinations thereof; and at least one triprotic acid, the method comprising the steps of: a. coating at least a portion of the first component with the at least one triprotic acid; and/or b. absorbing into at least a portion of the first component the at least one triprotic acid.

Description

[0123] Embodiments of the present invention will now be further described with reference to the following non-limiting examples and accompanying figure in which:

[0124] FIG. 1: is a graph showing the results from testing using the MBEC Assay for Pseudomonas aeruginosa ATCC 13359;

[0125] FIG. 2: is a graph showing the results from testing using the MBEC Assay for Staphylococcus haemolyticus;

[0126] FIG. 3: is a graph showing the results from testing using the MBEC Assay for MRSA 308;

[0127] FIG. 4: is a graph showing the results from testing using the CDC Reactor Model for Pseudomonas aeruginosa ATCC 10434;

[0128] FIG. 5: is a graph showing the results from testing using the CDC Reactor Model for Staphylococcus haemolyticus NCTC 11042;

[0129] FIG. 6: is a graph showing the results from testing using the CDC Reactor Model for Methicillin-resistant Staphylococcus aureus 308;

[0130] FIG. 7: is a graph showing the results from testing using the CDC Reactor Model for Pseudomonas aeruginosa ATCC 10434;

[0131] FIG. 8: is a graph showing the results from testing using the CDC Reactor Model for Pseudomonas aeruginosa ATCC 10434;

[0132] FIG. 9: is a graph showing the results from testing using the CDC Reactor Model for Staphylococcus aureus;

[0133] FIG. 10: is a graph showing the results from testing using the CDC Reactor Model for Staphylococcus aureus;

[0134] FIG. 11: is a graph showing the quantity of viable Staphylococcus aureus recovered from a pre-formed 24 hour biofilm after 24 hour treatment with each agent;

[0135] FIG. 12: is a graph showing the quantity of viable Staphylococcus aureus recovered from a pre-formed 72 hour biofilm after 24 hour treatment with each agent.

[0136] General Method of Sample Preparation

[0137] General Method 1: First Component and Triprotic Acid

[0138] Triprotic acid (e.g. citric acid) powder was dissolved in de-ionised water and then mixed with non-aqueous solvent (e.g. IPA). The first component, typically in nonwoven fibre form, was placed into the triprotic acid solution and allowed to absorb the solution. The solution was then dried using thermal drying, to leave a solid chitosan, chitin or derivative thereof coated with the triprotic acid.

[0139] General Method 2: First Component with Triprotic Acid and Monoprotic Acid

[0140] Triprotic acid (e.g. citric acid) powder was dissolved in de-ionised water and then mixed with a solubilising acid (e.g. lactic acid) solution. This was then mixed with non-aqueous solvent (e.g. IPA). The first component, typically in nonwoven fibre form, was placed into the mixed acid solution and allowed to absorb the solution. The solution was then dried using thermal drying, to leave a solid chitosan, chitin or derivative thereof coated with a mixture of triprotic acid and solubilising acid.

EXAMPLE COMPOSITIONS

[0141] The following are examples of compositions prepared according to the present invention.

Example 1

[0142] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.439 g) to provide a nominal 32.5% composition.

Example 2

[0143] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.027-0.068 g) to provide a nominal 2-5% composition.

Example 3

[0144] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.27-0.41 g) to provide a nominal 20-30% composition.

Example 4

[0145] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.81-0.94 g) to provide a nominal 60-70% composition.

Example 5

[0146] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.027-0.068 g) and lactic acid (0.34 g) to provide a nominal 2-5% triprotic acid and 25% monoprotic acid composition.

Example 6

[0147] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.27-0.41 g) and lactic acid (0.34 g) to provide a nominal 20-30% triprotic acid and 25% monoprotic acid composition.

Example 7

[0148] 100% chitosan fibre woven (1.35 g) was coated with citric acid (0.81-0.94 g) and lactic acid (0.41 g) to provide a nominal 60-70% triprotic acid and 30% monoprotic acid composition.

Example 8

[0149] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.04 g) and lactic acid (0.2 g) to provide a nominal 3% triprotic acid and 15% monoprotic acid composition.

Example 9

[0150] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.34 g) and lactic acid (0.2 g) to provide a nominal 25% triprotic acid and 15% monoprotic acid composition.

Example 10

[0151] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.81 g) and lactic acid (0.27 g) to provide a nominal 60% triprotic acid and 20% monoprotic acid composition.

Example 11

[0152] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.41 g) and lactic acid (0.34 g) to provide a nominal 30% triprotic acid and 25% monoprotic acid composition.

Example 12

[0153] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.54 g) and lactic acid (0.34 g) to provide a nominal 40% triprotic acid and 25% monoprotic acid composition.

Example 13

[0154] 100% chitosan fibre nonwoven (1.35 g) was coated with citric acid (0.54 g) and lactic acid (0.41 g) to provide a nominal 40% triprotic acid and 30% monoprotic acid composition.

Example Wound Dressings

Example 14

[0155] A composition of the present invention is prepared containing nominally 2 g 100% chitosan fibre nonwoven in 8 g de-ionised water with 0.6g citric acid. The composition was coated onto the surface of a non-reticulated polyurethane foam and allowed to dry. The foam containing the dried composition of the present invention was then adhesive bonded to a polyurethane foam, distal to a bonded polyurethane film layer, to form a wound contact layer.

Example 15

[0156] A composition of the present invention is prepared containing nominally 2 g 100% chitosan fibre nonwoven in 8 g de-ionised water with 0.6 g citric acid. The composition was coated onto the surface of a woven viscose textile and allowed to dry.

[0157] MBEC Assay 1

[0158] To determine the efficacy of antimicrobials against biofilms of various microorganisms, an MBEC (Minimum Biofilm Eradication Concentration) Assay was used.

[0159] The MBEC Assay is a high throughput screening assay used to determine the efficacy of antimicrobials against biofilms of various microorganisms. The MBEC Biofilm Inoculator consists of a plastic lid with 96 pegs and a corresponding base. There are two types of bases that may be used with the MBEC lid. One base contains 96 individual wells. The individual wells allow for the growth of a variety of microorganisms on the same peg lid. The other type of base is a corrugated through base that can contain only a single microorganism. Biofilms are established on the pegs under batch conditions (no flow of nutrients into or out of an individual well) with gentle mixing. The established biofilm is transferred to a new 96 well plate for antimicrobial efficacy testing. The assay design allows for the simultaneous testing of multiple biocides at multiple concentrations with replicate samples, making it an efficient screening tool.

[0160] Test Microorganisms

[0161] Pseudomonas aeruginosa ATCC 13359

[0162] Staphylococcus haemolyticus

[0163] MRSA 308

[0164] Samples Tested

[0165] Control: Phosphate Buffered Saline (PBS)

[0166] Sample A: 100% chitosan fibre nonwoven with Ag (nominally 1%) and lactic acid to provide a nominal 25% monoprotic acid composition (comparative sample)

[0167] Sample B: Carboxymethylated cellulose fibre nonwoven with Ag (nominally 1%) (comparative sample—Aquacel Ag®)

[0168] Sample C: 100% Chitosan fibre nonwoven with lactic acid to provide a nominal 25% monoprotic acid composition (comparative sample)

[0169] Sample D: 100% Chitosan fibre nonwoven with acetic acid to provide a nominal 25% monoprotic acid composition (comparative sample)

[0170] Sample E: 100% Chitosan fibre nonwoven with citric acid to provide a nominal 25% triprotic acid composition (Example 3)

[0171] Preparation of Bacterial Inoculum

[0172] A 24 hour culture of each microorganism was harvested from either a Tryptone Soya Agar (TSA) plate or Brain Heart Infusion Agar (BHIA) plate and suspended in either 20 ml of Tryptone Soya Broth (TSB) or 20 ml of Brain Heart Infusion Broth (BHIB). The resultant bacterial suspension was diluted to give an initial OD590=0.10±0.03, which corresponds to a bacterial concentration of 10.sup.8 cfuml.sup.−1. This initial inoculum was serially diluted a further six times in order to represent progressively lower bacterial loads (i.e. 10.sup.7, 10.sup.6, 10.sup.5, 10.sup.4, 10.sup.3 cfuml.sup.−1). The starting bacterial concentration for each organism was typically 1×10.sup.8±5×10.sup.7 cfuml.sup.−1.

[0173] MBEC Assay

[0174] The biofilms of each microorganism were grown on pin lid projections of a microtitre plate for 48 hours at 37° C., 50 rpm. After 48 hours the pin lids were removed, washed briefly in Phosphate Buffered Saline (PBS) to remove planktonic bacteria and then placed in the agent challenge plate for 24 hours. For the preparation of challenge plate for the wound dressings, 1 cm.sup.2 pieces were cut using sterile scissors and placed into the designated wells of a microtitre plate. The challenge plate for the granule test agents was prepared by weighing out 30 mg±3 mg of each granule formulation into the wells of a microtitre plate. The wound dressings and the granules were then activated with 150 μl of PBS. Following treatment, the pin lid projections were washed twice in PBS, then transferred into 200 μl of neutraliser and placed in a sonic water-bath for 5 minutes in order to recover remaining attached bacteria. Serial dilutions were carried out on the resultant recovery broth and drop plates were used to quantify the recovered bacteria. All samples were tested in triplicate unless otherwise stated.

[0175] The results are shown in Table 1 and FIGS. 1 to 3. The results in FIGS. 1 to 3 relate to Samples B, C and E.

[0176] It is clear from the graphs shown in FIGS. 1 to 3 that Sample E comprising chitosan coated with a triprotic acid is effective against all three microorganisms tested. Sample C comprising chitosan fibres coated with a monoprotic acid showed microorganism concentrations for all three microorganisms tested. Finally, Sample B comprising carboxymethylated cellulose with silver showed good results for Staphylococcus haemolyticus but was not effective against either Pseudomonas aeruginosa ATCC 13359 and MRSA 308.

TABLE-US-00001 TABLE 1 Test results following MBEC Assay Organisms P. aeruginosa P. aeruginosa E. faecillis Sample (1 strain) (2 strain) (VRE) MRSA S. epidermidis S. haemolyticus A Comparable Comparable >Log 4 Ranged >Log 4 >Log 4 to untreated to untreated between Log2 control control and > log 4 B ≦Log 3 Comparable Comparable Ranged Log 2 >Log 4 to untreated to untreated between Log2 control control and > log 4 C Comparable ≦Log 2 >Log 4 Ranged >Log 4 >Log 4 to untreated between Log2 control and > log 4 D >Log 4 ≦Log 3 >Log 4 Ranged >Log 4 ≦Log 2 between Log2 and > log 4 E >Log 4 >Log 4 >Log 4 >Log 4 >Log 4 >Log 4

[0177] CDC Reactor Model 1

[0178] To determine the biofilm removal capabilities of seven wound dressings, against three bacterial species, using a CDC reactor method.

[0179] Test Microorganisms

[0180] Staphylococcus haemolyticus NCTC 11042

[0181] Pseudomonas aeruginosa ATCC 10434

[0182] Methicillin-resistant Staphylococcus aureus 308

[0183] Samples Tested

[0184] Control: Phosphate buffered saline (PBS)

[0185] Sample F: 100% Chitosan fibre nonwoven with 15% lactic acid and 3% citric acid. This equates to 1.35 g chitosan, 0.2 g lactic acid and 0.04 g citric acid (Example 8).

[0186] Sample G: 100% Chitosan fibre nonwoven with 15% lactic acid and 25% citric acid. This equates to 1.35 g chitosan, 0.2 g lactic acid and 0.34 g citric acid (Example 9).

[0187] Sample H: 100% Chitosan fibre nonwoven with 20% lactic acid and 60% citric acid. This equates to 1.35 g chitosan, 0.27 g lactic acid and 0.81 g citric acid (Example 10).

[0188] Sample I: 100% Chitosan fibre nonwoven with 3% citric acid. This equates to 1.35 g chitosan and 0.04 g citric acid (Example 2).

[0189] Sample J: 100% Chitosan fibre nonwoven with 25% citric acid. This equates to 1.35 g chitosan and 0.34 g citric acid (Example 3).

[0190] Sample K: 100% Chitosan fibre nonwoven with 60% citric acid. This equates to 1.35 g chitosan and 0.81 g citric acid (Example 4).

[0191] The samples were prepared following the methods described hereinbefore.

[0192] Dressing samples were cut into approximately 1.5 cm.sup.2 pieces before use. Phosphate Buffered Saline (PBS) was used as the control.

[0193] Preparation of Bacterial Inoculum

[0194] A 24 hour culture of the test bacteria was harvested from a Tryptone Soya Agar (TSA) plate using a sterile swab and re-suspended in 20 ml of Tryptone Soya Broth (TSB). The bacterial suspension was diluted to give an OD590=0.10±0.03 which corresponds to a bacterial concentration of 108±5×10.sup.7 cfuml.sup.−1. This was further diluted in TSB and used as the inoculum for the CDC reactor containing the test coupons. The CDC reactor was incubated for 48 hours at 37° C., shaking at 50rpm in order to encourage biofilm growth.

[0195] Biofilm Treatment

[0196] After 48 hours the test coupons were removed from the CDC reactor and washed 3 times in sterile PBS in order to remove planktonic bacteria. The washed coupons were then treated by sandwiching the coupon between two 1.5 cm.sup.2 dressing samples. The dressings were activated prior to testing by the addition of 350 μl PBS (75% saturation) to each 1.5 cm.sup.2 piece. Control coupons were submerged in 2 ml of PBS for P. aeruginosa (or in the case of S. haemolyticus and MRSA in 2 ml PBS+0.1% TSB). All samples were tested in triplicate. Microorganisms were recovered from the coupons after 24 hours treatment and quantified by performing serial dilutions and drop plates.

[0197] The results are shown in FIGS. 4 to 6.

[0198] It is clear from the graphs shown in FIGS. 4 to 6 that Samples G, H and K are effective against all three microorganisms tested. Samples G and H comprise chitosan fibres coated with a triprotic acid and a monoprotic acid. Sample K comprises a chitosan fibre coated with a higher amount of a triprotic acid. Whilst Samples F, I and J were effective against methicillin-resistant Staphylococcus aureus 308, they were less effective against both Staphylococcus haemolyticus NCTC 11042 and Pseudomonas aeruginosa ATCC 10434.

[0199] The results of the CDC Reactor Model show that a chitosan coated with increasing amounts of triprotic acid, e.g. around 60%, is more effective against microorganisms than a lower amount of around 25% or less. Also, the results show that including a monoprotic acid with a triprotic acid can be effective in reducing the amount of triprotic acid required, e.g. reducing the amount of triprotic acid to around 25%.

[0200] CDC Reactor Model 2

[0201] To determine the biofilm removal capabilities of six wound dressings, against two bacterial species, using the CDC reactor method.

[0202] Test Microorganisms

[0203] Staphylococcus haemolyticus NCTC 8325

[0204] Pseudomonas aeruginosa ATCC 10434

[0205] Samples Tested

[0206] Control: Phosphate buffered saline (PBS)

[0207] Sample L: 100% Chitosan fibre nonwoven with 25% lactic acid and 30% citric acid. This equates to 1.35 g chitosan, 0.34 g lactic acid and 0.41 g citric acid (Example 11).

[0208] Sample M: 100% Chitosan fibre nonwoven with 25% lactic acid and 40% citric acid. This equates to 1.35 g chitosan, 0.34 g lactic acid and 0.54 g citric acid (Example 12).

[0209] Sample N: 100% Chitosan fibre nonwoven with 30% lactic acid and 40% citric acid. This equates to 1.35 g chitosan, 0.41 g lactic acid and 0.54 g citric acid (Example 13).

[0210] Sample O: 100% Chitosan fibre nonwoven.

[0211] Sample P: 55% Chitosan fibre/45% Viscose fibre nonwoven with 25% lactic acid. This equates to 0.74 g chitosan fibre, 0.61 g Viscose fibre and 0.34 g lactic Acid

[0212] Sample Q: Carboxymethylcellulose nonwoven with ionic silver-containing, antibiofilm formulation.

[0213] The samples were prepared following the methods described hereinbefore.

[0214] Dressing samples were cut into approximately 1.5 cm.sup.2 pieces before use. Phosphate Buffered Saline (PBS) was used as the control.

[0215] Preparation of Bacterial Inoculum

[0216] A 24 hour culture of each microorganism was harvested from a Tryptone Soya Agar (TSA) plate and resuspended in 20 ml of Tryptone Soya Broth (TSB). The resultant bacterial suspension was diluted to give an initial OD.sub.590=0.10±0.03, which corresponds to a bacterial concentration of 10.sub.8±5×10.sub.7 cfuml.sub.−1. This was further diluted to approximately 10.sub.7 cfuml.sub.−1 in TSB and was used as the initial inoculum for the CDC reactor. The CDC reactor was incubated for 24 and 72 hours at 37° C., shaking at 50 rpm in order to encourage biofilm growth.

[0217] Biofilm Treatment

[0218] After 24 hours and 72 hours the test coupons were removed from the CDC reactor and washed 3 times in sterile Phosphate Buffered Saline (PBS) in order to remove planktonic bacteria. The washed coupons were then treated by sandwiching each coupon between two pieces of wound dressing material. The dressings were activated prior to testing by the addition of 350 μl PBS containing 1% TSB. Control coupons were submerged in 2 ml of PBS containing 1% TSB. Following the 24 hour treatment period the coupons were placed in 2 ml of neutraliser and sonicated for 15 minutes to recover remaining attached bacteria. Serial dilutions were carried out on the resultant recovery broth and drop plates were used to quantify the recovered bacteria. All samples were tested in triplicate.

[0219] The results are shown in FIGS. 7 to 10.

[0220] FIG. 7 shows the quantity of viable Pseudomonas aeruginosa recovered from 24 hour pre-formed biofilms following 24 hours treatment with the sample wound dressings. Controls were treated with PBS+1% TSB.

[0221] FIG. 8 shows the quantity of viable Pseudomonas aeruginosa recovered from 72 hour pre-formed biofilms following 24 hours treatment with the sample wound dressings. Controls were treated with PBS+1% TSB.

[0222] FIG. 9 shows the quantity of viable Staphylococcus aureus recovered from 24 hour pre-formed biofilms following 24 hours treatment with the sample wound dressings. Controls were treated with PBS+1% TSB.

[0223] FIG. 10 shows the quantity of viable Staphylococcus aureus recovered from 72 hour pre-formed biofilms following 24 hours treatment with the sample wound dressings. Controls were treated with PBS+1% TSB.

[0224] It is clear from the graphs shown in FIGS. 7 to 10 that Samples L, M and N of the present invention are effective against both microorganisms tested. Whilst Samples Q and P were effective against Staphylococcus aureus, they were less effective against Pseudomonas aeruginosa ATCC 10434.

[0225] MBEC Assay 2

[0226] Test microorganism

[0227] Staphylococcus aureus NCTC 8325

[0228] Agents Tested

TABLE-US-00002 Control Phosphate Buffered Saline (PBS) + 1% Tryptone Soya Broth 1 2.5% Chitosan with 50% citric acid and 25% lactic acid 2 Chitosan with 30% citric acid and 25% lactic acid 3 Chitosan with 50% citric acid and 25% lactic acid 4 Chitosan with 15% citric acid and 25% lactic acid 5 Chitosan with 50% citric acid

[0229] All test agents were prepared by coating chitosan granules having a degree of deacetylation >75% with the specified acid(s) using a non-aqueous solvent. This formed a solid chitosan salt in the form of a granule. The granules were then mixed into a gel formulation using de-ionised water with the gel containing between 5-10% chitosan salt.

[0230] MBEC Assay

[0231] The S. aureus biofilm was grown on pin lid projections of a microtitre plate for 24 hours and 72 hours at 37° C. After 24 hours and 72 hours the pin lids were removed, washed briefly in sterile Phosphate Buffered Saline (PBS) to remove planktonic bacteria and then placed in the agent challenge plate for 24 hours. For the preparation of the challenge plate an amount of each gel formulation was placed into the wells of a microtitre plate. Positive and negative control pin lid projections were placed in PBS+1% TSB. Following treatment, the pin lid projections were washed twice in PBS and then placed in a neutraliser. Plates were sonicated. Serial dilutions were carried out on the resultant recovery broth and drop plates were used to quantify the recovered bacteria. All samples were tested in triplicate.

[0232] Twenty-Four Hour Biofilm

[0233] All the agents tested successfully reduced the number of viable bacteria recovered from within the pre-formed 24 hour biofilms of S. aureus after treatment for 24 hours at 37° C., as shown in FIG. 11. This represented a 5.84±0.53 log reduction in viable bacteria compared with the untreated controls.

[0234] Seventy-Two Hour Biofilm

[0235] All the agents tested successfully reduced the number of viable bacteria recovered from within the pre-formed 72 hour biofilms of S. aureus after treatment for 24 hours at 37° C., as shown in FIG. 12. For each agent this represented a 5.52±0.22 log reduction in viable bacteria compared with the untreated controls.

[0236] It is of course to be understood that the present invention is not intended to be restricted to the foregoing examples which are described by way of example only.