Antimicrobial composition

Abstract

An antiseptic composition suitable for use on skin and wounds comprising a source of an antimicrobial agent and an agent which disrupts biofilms. More, particularly the invention relates to a composition capable of providing effective antimicrobial activity while at the same time avoiding wound and skin irritation and retardation of wound healing.

Claims

1. An antiseptic composition suitable for use on skin and wounds comprising a source of an antimicrobial agent that is silver, a buffering agent, wherein the pH is maintained at a pH of between 4 and 6, and EDTA or its salts as an agent which disrupts biofilms.

2. The antiseptic composition as claimed in claim 1 wherein the EDTA is in the form of the di-, tri- or tetra-basic salts of EDTA.

3. The antiseptic composition as claimed in claim 1 wherein the composition has a pH of between 4.5 and 5.5.

4. The antiseptic composition as claimed in claim 1 wherein the composition is in the form of a gel.

5. The antiseptic composition as claimed in claim 1 wherein the antimicrobial agent is ionic silver.

6. The antiseptic composition as claimed in claim 5 wherein the composition comprises from about 0.1% to about 10% by weight of ionic silver.

7. The antiseptic composition as claimed in claim 6 wherein the composition comprises from about 0.5% to about 1.5% by weight of ionic silver.

8. A method of making an antiseptic composition as claimed in claim 2 comprising the steps of adding EDTA as a tetrasodium salt in aqueous solution to an antiseptic composition comprising a source of an antimicrobial agent that is silver and then adjusting the pH of the composition to a pH of between 4 and 6.

9. A method of treating chronic wounds or burns comprising applying to a chronic wound or a burn the composition of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the antimicrobial activity of a two component iodine generating solution with and without EDTA against Staphylococcus aureus.

(2) FIG. 2 is a graph showing zone of inhibition with EDTA.

(3) FIG. 3 is a graph showing zones of inhibition with EDTA, with and without poloaxmer.

DETAILED DESCRIPTION OF THE INVENTION

(4) The compositions according to a first aspect of the invention comprise an antiseptic agent, preferably molecular iodine, ionic silver, chlorhexidine, or hydrochloric acid or an oxidizing agent such as sodium hypochlorite, chlorine dioxide, hydrogen peroxide or peroxy acid salts. The antiseptic agent is preferably included in the composition at a level of from 0.01% to 10% by weight, more preferably 0.1% to 5% by weight. Iodine is preferably included in the composition at a level of from 0.01% to 10% by weight and more preferably from 0.1% to 1.0% by weight. Preferably, the source of iodine is an iodide and the composition further comprises an oxidant and a buffer, the oxidant being held separately from the iodide until the point of use. The buffer is preferably capable of maintaining the pH of the composition at between 4.5 and 6 so that iodine is generated at a physiologically acceptable and efficacious rate. Compositions comprising iodide and an oxidant held separately from the iodide are described further in EP1158859B.

(5) Where iodide is present, the amount of oxidant in the composition is tailored to provide a stoichiometric match with iodide. Preferably, the oxidant is iodate and is provided in a molar ratio of from 1:4 to 1:10 with iodide. In this way, the iodide present in the composition fully reacts with the oxidant. Iodide and iodate are preferably present as sodium salts although other usual counter ions may be present.

(6) Where the antiseptic agent is ionic silver, it is preferably included in the 10 composition at a level of from 0.1% to 10% by weight and more preferably 0.5% to 1.5% by weight.

(7) The pH of the composition is generally below 8 and preferably between 4 and 8, more preferably between 4 and 6 and most preferably between 4.5 and 5.5. The desired pH may be achieved by incorporating buffering agents in the composition. Examples of buffering agents which may be included are citric acid/di-sodium hydrogen phosphate, citric acid/sodium citrate, acetic acid/sodium acetate. The buffering agent may conveniently be present in an amount of about 1% to 20% by weight of the composition, preferably about 4% to 6% by weight and more preferably about 5% by weight so as to provide an isotonic composition

(8) EDTA is preferably present as the di-, tri- or tetra-basic salts of EDTA. We have found that these salts are effective for eradicating microorganisms in the free floating or planktonic state and biofilm state alone or in the presence of an antiseptic agent. For example, we have found that EDTA at concentrations of 0.1-40% weight by volume was effective in killing a range of microorganisms both in the planktonic and biofilm state. Microorganisms that were effectively killed by EDTA included Pseudomonas aeruginosa, Serratia marcescens, vancomycin resistant Enterococcus (VRE) and methicillin resistant Staphylococcus aureus (MRSA).

(9) EDTA is preferably present in the compositions of either aspect of the present invention at a level of 0.5% to 10% by weight of the composition, more preferably 1% to 3% by weight.

(10) The compositions of the present invention may be in the form of a water based gel which maintains a moist wound healing environment and promotes healing. A gel gives the advantage of flow into the wound to form an intimate contact with the wound bed and provide antimicrobial effects to the whole wound. Preferably, the gel has a high enough viscosity that it does not flow out of wounds on areas of the body that are or become non-horizontal. Preferably, the pH of the gel is buffered at around 5.5 as this does not alter the pH balance of the peri-wound tissue and, therefore, protects it.

(11) The following examples are illustrative of the present invention.

Example 1

(12) Effect of EDTA on Iodine

(13) Compositions containing iodine and EDTA were prepared by making a pair of aqueous gels (see Table 1 for Gel A and Table 2 for Gel B) which were intimately mixed at the point of use. Each gel was made by preparing an aqueous solution containing all of the appropriate water-soluble parts according to the formulations below and then adding a slurry of a non-ionic cellulosic viscosifier (hydroxyethylcellulose) in propylene glycol.

(14) TABLE-US-00001 TABLE 1 Component % w/w Water To 100 Propan-1,2-diol 10 Hydroxyethylcellulose 3.86 Sodium iodate 0.16 Citric acid 3.99 di-sodium phosphate 15.06

(15) TABLE-US-00002 TABLE 2 Component % w/w Water To 100 Propan-1,2-diol 10 Hydroxyethylcellulose 4.14 Sodium iodide 0.59 Ethylenediaminetetraacetic 0.1-4.0 acid tetra-sodium salt tetrahydrate Phosphoric acid As necessary to adjust aqueous phase to ph 5.5

(16) A commercially available cadexomer iodine ointment was present as a positive control as it contains 0.9% iodine. Intrasite is an amorphous hydrogel and was present as a negative control as it contains 0% iodine.

(17) 9 ml of simulated wound fluid was added to a 17 ml volume cell well. A 1 ml culture of Staphylococcus aureus was added to each well to give a final culture concentration of 106 cfu/ml. Controls for this experiment involved adding 2 g of cadexomor iodine ointment (positive control) and nydrogel (negative control) separately to three cell wells each. 1 g of gels A and B, with different concentrations of EDTA, were then added to separate cell wells (in triplicate). The cell wells containing the culture and gels were then shaken at 600 rpm at 35 C. After time intervals of 4, 24, 48, 72 and 96 hours, a 0.1 ml test sample was taken from each well and placed into 9.9 ml MRD (maximum recovery diluent) containing 1% sodium thiosulphate. A 1 ml sample was then transferred to Tryptone Soy Agar plates and incubated for 48 hours. Bacterial counts were then recorded.

(18) TABLE-US-00003 TABLE 3 Test 4 24 48 72 96 composition hours hours hours hours hours lodosorb(positive 10 10 10 10 10 control) Intrasite(negative 7600000 310000000 530000000 1000000000 14000000 control) 0% 1.sub.2 7600000 310000000 530000000 1000000000 14000000 0% EDTA 0.3/01.sub.2 1240000 1000000 1000000 10000000 100000000 0% EDTA 0.3/01.sub.2 1010000 1000000 1000000 10000000 100000000 0% EDTA 0.3/01.sub.2 1670000 1000000 1000000 10000000 100000000 0% EDTA 0.3% 1.sub.2 1500000 1000000 1000000 1000000 1000000 0.5% EDTA 0.3% 1.sub.2 92000 10 10 10 10 0.5% EDTA 0.3% 1.sub.2 58000 10 10 10 10 0.5% EDTA 0.3.sup.1/01.sub.2 8100 10 10 10 10 1% EDTA 0.3.sup.1/01.sub.2 130000 10 10 10 10 1% EDTA 0.3.sup.1/01.sub.2 110000 10 10 10 10 1% EDTA 0.3% 1.sub.2 710 10 10 10 10 1.75% EDTA 0.3% 1.sub.2 4400 10 10 10 10 1.75% EDTA 0.3% 1.sub.2 31000 10 10 10 10 1.75% EDTA 0.3% 1.sub.2 8500 10 10 10 10 2.5% EDTA 0.3% 1.sub.2 10600 10 10 10 10 2.5% EDTA 0.3% 1.sub.2 6200 16000 10 10 10 2.5% EDTA 0.3.sup.1/01.sub.2 93000 10 10 10 10 4% EDTA 0.3.sup.1/01.sub.2 8500 10 10 10 10 4% EDTA 0.3.sup.1/01.sub.2 1400 10 10 10 4% EDTA

(19) The results shown in Table 3 are also shown graphically in FIG. 1.

(20) These results show that by adding as little as 0.5% EDTA (calculated as the di-sodium salt) to iodine, the efficacy of iodine, at 0.3%, is enhanced when compared to the control of 0.3% iodine with no EDTA. Clearly, from the results, EDTA enhances the effects of iodine within 24 hours to the same efficacy of 0.9% iodine (positive control).

Example 2

(21) Zones of Inhibition With Tetra Sodium EDTA

(22) Seven antibiotic resistant microorganisms were used to evaluate the efficacy of EDTA in killing bacteria and yeasts grown on agar. For this experiment, filter paper discs were soaked in EDTA at concentrations ranging from 0.1-40%. EDTA was made up by dissolving it in an appropriate amount of sterile double distilled water. The filters were then added onto Muller Hinton agar which had been inoculated with a microorganism under study for 24 hours at 35 C. All microorganisms were tested twice.

(23) The results in FIG. 2 show that the zones of inhibition generally ranged from about 20 to 43 mm (including disc 13 mm). Zones were higher with increasing levels of EDTA indicating that EDTA alone is an effective antiseptic agent.

Example 3

(24) Zones of Inhibition With EDTA

(25) Poloxamer F127 hydrogels (Univar, Basildon, Essex, UK) are di-block co-polymers of polyoxyethylene and polyoxypropylene that demonstrate thermo-reversible gelation properties. At temperatures below 15 C., poloxamer is liquid and fully miscible with water but changes to a firm gel at temperatures in excess of 15 C. Poloxamer encourages bacteria to exhibit a more clinically relevant biofilm phenotype. In The Use of Poloxamer Hydrogels for the Assessment of Biofilm Susceptibility Towards Biocide Treatments. Gilbert P., Jones, M. V., Allison, D. G., Heys, S., Maira, T., Wood, P. Journal of Applied Microbiology (1998; 85:985-990), Gilbert, et al., determined that P. aeruginosa cells grown on poloxamer hydrogel (biofilm form) express outer membrane proteins between 78 and 87 kDa, which are not evident in cells grown on standard nutrient agar (planktonic). Consequently, poloxamer gel cultures mimic many of the properties of biofilm-grown P. aeruginosa (Gilbert et al., 1998). This indicates that there is a phenotypic difference between P. aeruginosa cells grown on poloxamer hydrogel and nutrient agar, with only poloxamer grown cells resembling biofilm cells. It was found from Wirtanen's study (Performance Evaluation of Disinfectant Formulations Using Poloxamer-hydrogel Biofilm-Constructs. Wirtanen, G., Salo, S., Allison, D. G., Mattila-Sandholm, T., Gilbert, P. Journal of Applied Microbiology (1998; 85:965-971)) that bacteria which are grown in poloxamer have biofilm properties and associated enhanced biocide resistance. Gilbert and colleagues suggested that bacteria grown in poloxamer hydrogels could be exposed to biocides to provide a reproducible method for testing the antimicrobial efficacy of biocides against biofilm bacteria (Gilbert, et al., 1998).

(26) Seven antibiotic resistant microorganisms were used to evaluate the efficacy of EDTA in killing bacteria and yeasts grown on poloxamer gel (biofilm state). For this experiment, filter paper discs were soaked in EDTA at concentrations ranging from 0.1-40%. In this present study, poloxamer F127, a di-block copolymer of polyoxyethylene and polyoxypropylene, was used as a medium on which bacteria could be grown as a biofilm phenotype and express the characteristics more appropriate to the real world. The filters were then added onto Muller Hinton agar which had been inoculated with the microorganism under study for 24 hours at 35 C. All plates were done in duplicate.

(27) The results in FIG. 3 show that the zones of inhibition were slightly smaller in the presence of poloxamer indicating that bacteria/yeasts growing as biofilms are physically more resistant to EDTA when compared to their planktonic counterparts. C kruzei and Ps aeruginosa were the exception with larger zones with poloxamer. Zones of inhibition were evident on all organisms tested at 40% EDTA. At 10% EDTA and 5% EDTA there were no zones with Serratia marcescens. At 0.1% EDTA no zones of inhibition were evident with any organism studied indicating that EDTA is not effective at this level.

Example 4

(28) Minimum Inhibitory Concentrations (MIC's) and Minimum Bactericidal Concentrations (MBC's) With Tetra, Tri and Di Basic Salts of EDTA

(29) A microtitre plate and optical density readings and visual inspection were performed to obtain the MIC's for a variety of microorganisms (see Table 4). Concentrations of EDTA included in this study were 40 mg/ml pH 10.00 as tetra-Na, 40 mg/ml as tri-Na pH 6.84, 40 mg/ml as di-Na pH 5.50. Into each microtitre plate, 100 pl of inoculum and EDTA was added. The plate was then incubated for 24 hours at 35 C.3 C. Following incubation all microtitre plates were inspected visually for growth.

(30) The results are presented in Table 4.

(31) TABLE-US-00004 TABLE 4 PvilIC, .sup.s MICRO IDENTIFI- INOCU- di tri tetra ORGANISM CATION LUM cfu Na Na Na S marcescens multi res 5.00E+04 0.938 1.875 0.938 Ps aeruginosa NCTC 8506 6.75E+04 0.234 0.234 0.234 (res) Ps aeruginosa NCIMB 8626 7.00E+04 0.938 0.938 0.938 E coli NCIMB 8545 5.50E+04 0.469 0.469 0.469 E coli NCIMB 10544 <5.0+04.sup. 0.469 0.938 0.469 KI 033 clinical <5.0+E4 0.938 0.938 1.875 pneumoniae isolate Ent cloacae 166 clinical 5.00E+04 1.875 0.938 0.938 isolate Pr mirabilis NCTC 9559 1.50E+05 0.469 0.469 0.469 A baumannii NCIMB 9214 4.40E+04 0.234 0.234 0.469 S aureus NCIMB 9518 4.00E+04 0.234 0.234 0.234 MRSA 1 Cardiff PHL 5.50E+04 0.117 0.469 0.234 MRSA 2 Cardiff PHL 5.75E+04 0.234 0.469 0.234 MRSA 26 1.25E+04 0.234 0.234 0.234 MRSA NCTC 12232 5.00E+04 0.234 0.234 0.469 MRSA NCTC 10442 6.50E+04 0.234 0.234 0.234 MRSA 103731 Chester 6.00E+04 0.234 0.117 0.234 PHL Ent faecalis 141 clinical 2.50E+04 0.234 0.234 0.234 isolate VRE 1 Cardiff PHL 2.75E+04 0.234 0.469 0.234 VRE 2 Cardiff PHL 3.25E+04 0.234 0.234 0.234 VRE NCTC 12201 6.00E+04 0.234 0.234 0.234 Strep NCTC 8198 7.00E+04 0.234 0.469 0.469 pyogenes B subtilis NCTC 3610 3.00E+04 0.234 0.234 0.234 C krusei NCPF 3876 1.50E+04 0.938 0.938 0.938 (res) B fragilis NCIMB 9343 1.90E+05 0.117 0.234 0.234 C/perfringens 362 clinical 9.00E+03 0.117 <0.117 <0.117 isolate Pep NCTC 11460 1.30E+06 0.234 0.469 0.469 anaerobius

(32) In general all MIC's recorded were equivalent for all the salts of EDTA studied. This suggests that the pH of the solutions is equivalent. Therefore, the activity of EDTA is not affected by the salt form added to the microtitre plate. These results show that low concentrations of EDTA are very effective on bacteria.

Example 5

(33) The Effect of EDTA on the Antimicrobial Efficacy of Silver Containing Wound Dressings

(34) The antimicrobial dressings used in this study were Acticoat (Smith and Nephew) and AQUACEL Ag Hydrofiber (ConvaTec). Acticoat is a nanocrystalline silver antimicrobial barrier dressing which consists of a rayon/polyester non-woven inner core laminated between two layers of silver-coated high density polyethylene mesh (HDPE). The layers are held together with ultrasound welds. AQUACEL Ag is comprised of sodium carboxymethylcellulose Hydrofiber and ionic silver. The silver cations in AQUACEL Ag are associated with the individual highly absorbent anionic carboxymethylcellulose fibres of the Hydrofiber dressing. AQUACEL Hydrofiber dressing (without silver) was also used as a control.

(35) All dressings (AQUACEL Hydrofiber dressing [controlwithout silver], AQUACEL Ag and Acticoat (nanocrystalline dressing) were hydrated with 20 mg/ml of tetra-Na EDTA. All tests were performed against Ps aeruginosa and tested on Mueller Hinton agar (MHA) and Poloxamer gel (incorporating Mueller Hinton broth (MHB). This involved inoculating either a MH agar plate or poloxamer gel plate with a specific isolate and then adding an appropriate hydrated (to saturation point) wound dressing (360 I (MRD) for AQUACEL and AQUACEL Ag and 150 I (sterile distilled water- as per manufacturers instructions) for Acticoat). The plates were then incubated at 35 C.3 C. for 24 hours after which the zone of clearance (no growth) around the dressing was measured. Zones of inhibition were measured horizontally and vertically (inclusive of the dressing sample) and a mean value was calculated from the duplicate set of results. The mean dressing size was then subtracted from the mean zone of inhibition to determine the corrected zone of inhibition (CZOI). A CZOI test allows for any inherent variability in the shape and size of zones created by the silver dressings which may change in dimension following hydration.

(36) Table 5 shows the size of corrected zone of inhibition (CZOI) in mm on MH agar and poloxamer gel.

(37) TABLE-US-00005 TABLE 5 MHA Poloxamer gel with MHB (Non-biofilm bacteria) (biofilm bacteria) Hydrating AQUACEL AQUACEL Fluid AQUACEL Ag Acticoat AQUACEL Ag Acticoat EDTA 0 16 10 6 6 7 MRD 0 18 N/A 0 3 N/A Water N/A N/A 7 N/A N/A 3

(38) When EDTA was added to AQUACEL, no zones of inhibition were observed on MHA (non-biofilm state). However, ZOI's were observed around AQUACEL Ag on MHA (non-biofilm bacteria) when EDTA or (maximal recovery diluent) MRD was added indicating the antimicrobial activity of silver. Larger ZOI's were observed around Acticoat following the addition of EDTA when compared to hydration with water. In the presence of poloxamer gel an increase in the CZOI was observed following hydration of AQUACEL Ag with MRD when compared to EDTA indicating an additive effect with the use of EDTA. Overall the results showed that EDTA enhances the effects of ionic silver on bacteria grown in the biofilm state (poloxamer gel). Overall these results suggest that by using an EDTA dressing it is effective against both planktonic and biofilm microorganisms.

(39) Table 6 shows the size of corrected zone of inhibition in mm on MH agar and poloxamer gel.

(40) TABLE-US-00006 TABLE 6 MHA Poloxamer gel with MHB (non-biofilm bacteria) (biofilm bacteria) Nano- Nano- crystalline crystalline Hydrating AQUACEL silver AQUACEL silver fluid AQUACEL Ag dressing AQUACEL Ag dressing EDTA 0 15.99 9.57 6.28 5.96 6.73 MRD 0 18.04 N/A 0 3.42 N/A Water N/A N/A 6.74 N/A N/A 2.82

Example 6

(41) Minimum Inhibitory Concentrations (MIC) for a Two Component Gel Comprising Iodine and a Two Component Gel as Detailed in Example 1 Containing EDTA (diNa EDTA, triNa EDTA and tetraNa EDTA)

(42) This experiment shows the effect of pH on the activity of a range of EDTA forms.

(43) For this experiment a two component gel as detailed in Example 1 was made without the HEC component (gel). This experiment was therefore performed using only liquid compositions (see Table 7 for TCG Solution (A) and Table 8 for TCG Solution (B)) in order for MIC's to be calculated. The pH of the TCG/EDTA solutions was approx 5.5. The organisms tested included: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli (repeated twice) and C albicans.

(44) TABLE-US-00007 TABLE 7 Component % w/w Water To 100 Sodium iodate 0.1 Citric acid 3.99 di-sodium phosphate 15.06

(45) TABLE-US-00008 TABLE 8 Component % w/w Water To 100 Sodium iodide 0.4 Ethylenediaminetetraacetic As shown in Table 1 acid tetra-sodium salt tetrahyd rate Phosphoric acid As necessary to adjust aqueous phase to pH 5.5

(46) Table 9 shows MIC's for TCG solution and TCG solution combined with EDTA (values in brackets are EDTA concentrations in mg/ml)

(47) TABLE-US-00009 TABLE 9 TCG TCG and tri TCG and tetra Bacterium solution* TCG EDTA Na EDTA Na EDTA S aureus 0.25 (0) 0.008 (0.31) 0.008 (0.31) 0.008 (0.31) Ps aeruginosa 0.5 (0) 0.062 (2.5) 0.062 (2.5) 0.062 (2.5) E coli 0.25 (0) 0.031 (1.25) 0.031 (1.25) 0.031 (1.25) *(working concentration was 0.2% iodine)

(48) MIC's were reduced considerably in the presence of EDTA (di, tri and tetra Na). It can be seen that the MICs, expressed as Iodine concentration, are lower in the presence of di, tri and tetra sodium EDTA.

(49) The antimicrobial benefit of having EDTA present is achieved at concentrations of 0.31 mg/ml. Despite different forms of EDTA being known to provide differential antimicrobial efficacy, at a constant pH (5.5 in this case) all EDTA forms were equally effective in significantly reducing the MIC's for all three organisms compared to the iodine generating solution without EDTA.