Antimicrobial Compositions

Abstract

Liquid compositions are described which have enzyme that is able to convert a substrate to release hydrogen peroxide; substrate for the enzyme; and polymer. The substrate is less than 10% by weight of the composition and the composition does not comprise sufficient free water to allow the enzyme to convert the substrate, or has a water activity of 0.7 or less.

Claims

1. A composition comprising: enzyme that is able to convert a substrate to release hydrogen peroxide; substrate for the enzyme, the substrate being less than 10% by weight of the composition; and polymer, wherein the composition does not comprise sufficient free water to allow the enzyme to convert the substrate or has a water activity of 0.7 or less wherein the composition is a liquid.

2. A composition according to claim 1, wherein the substrate is at least 1%, by weight, of the composition, preferably at least 2%, by weight, of the composition.

3. A composition according to claim 1 or claim 2, comprising solute.

4. A composition according to claim 3, wherein the solute has a solubility of at least 300 g/100 g water at 20° C. and 1 atm.

5. A composition according to claim 3 or claim 4, wherein the solute is, or comprises, a sugar or sugar derivative.

6. A composition according to any of claims 3 to 5, wherein the solute is, or comprises, fructose.

7. A composition according to any preceding claim, wherein the solute is 50% or less, by weight, of the composition, preferably 45% or less by weight of the composition.

8. A composition according to any preceding claim, which does not comprise honey.

9. A composition according to any preceding claim, which comprises substantially no oil or lipophilic phase.

10. A composition according to any preceding claim, comprising non-aqueous solvent.

11. A composition according to claim 10, wherein the non-aqueous solvent is, or comprises, a polyol, preferably wherein the polyol is glycerol.

12. A composition according to claim 10 or claim 11, wherein the non-aqueous solvent is present in an amount of at least at least 5% by weight.

13. A composition according to any of claims 10 to 12, wherein the non-aqueous solvent is present in an amount of 50% or less, by weight, preferably 40% or less by weight.

14. A composition according to any preceding claim, comprising antioxidant.

15. A composition according to any preceding claim, wherein the antioxidant is ascorbic acid, tocopherol or ascorbyl palmitate, preferably ascorbic acid.

16. A composition according to claim 14 or claim 15, wherein the antioxidant is present in an amount of at least 0.05%, preferably at least 0.1%, by weight.

17. A composition according to any of claims 14 to 15, wherein the antioxidant is present in an amount of 2% or less by weight, preferably 1% or less by weight.

18. A composition according to any preceding claim, which comprises substantially no hydrogen peroxide.

19. A composition according to any preceding claim, wherein hydrogen peroxide is present at a concentration of 6 ppm or less.

20. A composition according to claim 19, wherein the hydrogen peroxide is present at a concentration of 3 ppm or less.

21. A composition according to any preceding claim, which is formulated such that at 24 hours, following a 1:1 dilution (by weight) with water, the level of hydrogen peroxide production increases by a factor of at least 200.

22. A composition according to any preceding claim, wherein the polymer is polyethylene glycol.

23. A composition according to any preceding claim, comprising a buffer.

24. A composition according to any preceding claim, which is at a pH of 5 to 7.5.

25. A composition according to any preceding claim, wherein the enzyme is glucose oxidase and the substrate for the enzyme is D-glucose.

26. A composition according to any preceding claim which is sterile.

27. A composition according to any preceding claim, comprising substantially no zinc oxide, catalase, and/or peroxidase.

28. A composition according to any preceding claim, wherein the enzyme is a purified enzyme, preferably wherein the enzyme has a mass purity at least 95%, preferably at least 98%.

29. A composition according to any preceding claim, wherein the substrate is a purified substrate, preferably wherein the substrate has a mass purity of at least 95%, preferably at least 98%.

30. A composition according to claim 3 or an claim dependent on claim 3, wherein the solute is a purified solute, preferably wherein the solute has a mass purity of at least 98%.

31. A composition according to any preceding claim, wherein the enzyme is 0.001% to 0.5%, by weight, of the composition or 0.0025% to 0.2%, by weight, of the composition.

32. A composition according to any preceding claim, which has a viscosity of 10000 to 20000 mPas at 20° C. and 1 atm, optionally 12000 to 15000 mPas at 20° C. and 1 atm.

33. A composition according to any preceding claim, with a water activity of 0.6 or less.

34. A wound dressing which comprises a dressing material for dressing a wound, and a composition according to any preceding claim.

35. A dispensing device containing a composition as defined in any of claims 1 to 33, optionally wherein the dispensing device is a spray, a tube or a syringe.

36. A composition according to any of claims 1 to 33, for use as a medicament.

37. A composition according to any of claims 1 to 33, for use in prevention, treatment or amelioration of a microbial infection.

38. A composition according to any of claims 1 to 33, for use in treatment of a wound.

39. A method of preparing a composition, the method comprising, combining: an enzyme that is able to convert a substrate to release hydrogen peroxide; a substrate for the enzyme; and polymer, wherein the composition is formulated to have a water activity of 0.7 or less, or such that it does not include sufficient free water to allow the enzyme to convert the substrate, wherein the composition is formulated such that it is a liquid, and wherein substrate is added such that it is less than 10% by weight of the composition.

40. A method according to claim 39, comprising adding non-aqueous solvent.

41. A method according to any of claims 39 to 40, comprising adding antioxidant.

42. A method according to any of claims 40 to 41, wherein the polymer is polyethylene glycol.

43. A method according to any of claims 39 to 42, wherein the composition is formulated to have a viscosity of 10000 to 20000 mPas at 20° C., optionally 12000 to 15000 mPas at 20° C. and 1 atm.

44. A method according to any of claims 39 to 43, wherein the enzyme is a purified enzyme, preferably with a mass purity of at least 95%.

45. A method according to any of claims 39 to 44, wherein the substrate is a purified substrate, preferably with a mass purity of at least 98%.

46. A method according to any of claims 39 to 45, comprising a solute.

47. A method according to claim 46, wherein the solute has a solubility of at least 300 g/100 g water at 20° C. and 1 atm.

48. A method according to claim 46 or claim 47, wherein the solute is a sugar or sugar derivative, preferably fructose.

49. A method according to any of claims 46 to 48, wherein the solute is a purified solute, preferably with a mass purity of at least 98%.

50. A method according to any of claims 39 to 49, comprising sterilisation such that the composition is sterile.

51. A method according to claim 50, comprising sterilising by irradiation, preferably using gamma irradiation or electron beam irradiation.

52. A method according to any of claims 39 to 51, wherein the enzyme is glucose oxidase and the substrate is glucose.

53. A method according to any of claims 39 to 52, wherein the composition is formulated such that hydrogen peroxide peroxide is present at a concentration of 6 ppm or less, 5 ppm or less or 3 ppm or less.

54. A method according to any of claims 39 to 53, comprising adding a buffer.

Description

[0250] Preferred embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

[0251] FIG. 1 shows a hydrogen peroxide production profile, following dilution, of a formulation comprising 31% glucose, 52% fructose, 16.9% water and 0.1% glucose oxidase;

[0252] FIG. 2 shows a hydrogen peroxide production profile, following dilution, of a formulation comprising 31% glucose, 52% fructose, 9.9% water, 7% glycerol and 0.1% glucose oxidase;

[0253] FIG. 3 shows a hydrogen peroxide production profile, following dilution, of a formulation comprising 5% glucose, 78% fructose, 16.9% water and 0.1% glucose oxidase;

[0254] FIG. 4 shows a hydrogen peroxide production profile, following dilution, of a formulation comprising 5% glucose, 83% fructose, 11.9% water and 0.1% glucose oxidase;

[0255] FIG. 5 shows the water activities of the formulations of FIGS. 1 to 4;

[0256] FIG. 6 shows levels of hydrogen peroxide in formulations comprising 5% glucose, 78% fructose, 9.9% water, 7% glycerol and 0.025% glucose oxidase, both with and without 0.5% ascorbic acid, over a period of 7 days;

[0257] FIG. 7 shows glucose oxidase activity levels of the formulations of FIG. 5, over a period of 7 days; and

[0258] FIG. 8 shows a hydrogen peroxide production profile, following dilution, of a formulation comprising 5% glucose, 78% fructose, 9.9% water, 7% glycerol and 0.025% glucose oxidase, with both 0.1% ascorbic acid and 0.1% ascorbic acid.

EXAMPLE 1

Methods

[0259] Samples were formulated as follows (all amounts are expressed as % by weight).

TABLE-US-00001 TABLE 1 Glucose Glucose Fructose Water Glycerol Oxidase Sample/ 31 52 16.9 0 0.1 formulation 1 Sample/ 31 52 9.9 7 0.1 formulation 2 Sample/ 5 78 16.9 0 0.1 formulation 3 Sample/ 5 83 11.9 0 0.1 formulation 4

Stability Study

[0260] On the first day of the study the samples were placed at 25° C. in the incubator. Each day of the study (day 1, 3, and 6) samples were taken from the incubator, mixed by stirring and then analysed for glucose oxidase activity (TM-601) and hydrogen peroxide levels (TM-781). All dilutions were performed gravimetrically to ensure the highest level of accuracy. In addition on day 1 and 6 each of the samples was activated by the addition of an equal mass of water and incubated at 37° C. for up to 72 hours. At specified times the level of hydrogen peroxide was determined by testing an aliquot of the incubated activated solution.

Glucose Oxidase Assay (TM-601)

[0261] Approximately 1 g of samples were weighed into a 15 ml tube and diluted with 9 mL of phosphate buffered saline (PBS). All weights were measured using a 5 place balance for calculation of actual dilutions. The samples were vortex mixed to ensure that the entire sample was solubilised and then diluted serially to give a dilution between 5,000 and 100,000 times for analysis. 50 1-JL of sample, standard curve point, blank or quality control was pipetted into a 96 well plate and the reaction started by addition of 50 1-JL of substrate. The change in absorbance at 570 nm was monitored over a half an hour time period and the rate of change of OD used to calculate the glucose oxidase activity by comparison with the standard curve.

Hydrogen Peroxide Assay (TM-781)

[0262] Approximately 0.3 g of samples were weighed into 1.5 ml microfuge tubes and diluted with and equal mass of 99% ethanol. The solutions were physically mixed using a sterile pipette tip prior to being centrifuged to remove precipitated material. The ethanol supernatant was diluted through a series of three dilutions (500 to 10,000 times) with PBS. 50 1-JL of sample, standard curve point or blank was pipetted into a 96 well plate and the reaction started by addition of 50 1-JL of substrate. After five minutes the absorbance at 570 nm was determined and used to produce the standard curve and quantify the level of hydrogen peroxide in each sample.

Activated Hydrogen Peroxide Level Determination

[0263] In order to determine the level of hydrogen peroxide when the formulation was activated by contact with water approximately 0.65 g of sample was mixed well with an equal mass of water and made homogeneous by vortex mixing. This solution was incubated at 37° C. for up to 72 hours. Periodically samples were taken and extracted with 99% ethanol to precipitate the protein and sugar. Then following a dilution with PBS the samples were analyzed for hydrogen peroxide level using the Amplex Red hydrogen peroxide assay (TM-781) as detailed above.

Results

[0264] FIGS. 1, 2, 3 and 4 show graphical representations of the change in hydrogen peroxide concentration with time following activation of samples 1, 2, 3 and 4, respectively, by addition of water.

[0265] It can be observed how a significant reduction in the level of glucose can still result in a composition that is able to produce hydrogen peroxide over an extended period of time following dilution.

[0266] It is noted that the water activity of all samples was less than 0.6 (See FIG. 5).

[0267] It is also noted that Sample 4 was visually biphasic with significant amounts of precipitated sugar.

[0268] From the stability study, the following changes in glucose oxidase activity were observed.

TABLE-US-00002 TABLE 2 % change in glucose % change in glucose oxidase activity oxidase activity (day 1 to day 3) (day 1 to day 6) Sample/Formulation 1 −9.55 −22.05 Sample/Formulation 2 −2.22 +1.78

[0269] These data indicates that inclusion of a non-aqueous solvent such as glycerol can reduce loss of glucose oxidase activity.

EXAMPLE 2

[0270]

TABLE-US-00003 TABLE 3 Glucose Fructose Water Glycerol Glucose Oxidase Sample 1 31 52 9.9 7 0.1 (Variant 1) Sample 2 5 78 9.9 7 0.1 (Variant 2) All amounts % by weight

Methods

[0271] In order to prepare a range of products containing different levels of hydrogen peroxide, the samples were mixed according to the tables below, with the masses being recorded to calculate the exact dilution being performed

Sample 1:

[0272]

TABLE-US-00004 TABLE 4 Target Mass of Mass of Dilution Actual Dilution [Gox] Sample variant Factor [Gox] number (%) (g) Description of sample (g) (times) (%) 1 0.05 20.59269 0.1% GoX variant _ 20.59868 2.0 0.0500 2 0.025 20.24319 0.05% (dilution1) 20.30243 2.0 0.0250 3 0.005 10.01538 0.025% (dilution2) 40.05496 5.0 0.0050 4 0.003 30.01160 0.005% (dilution 3) 22.10454 1.7 0.0029 5 0.001 10.24168 0.003% (dilution4) 20.12446 3.0 0.0010

Sample 2:

[0273]

TABLE-US-00005 TABLE 5 Target Mass of Mass of Dilution Actual Dilution [Gox] Sample variant Factor [Gox] number (%) (g) Description of sample (g) (times) (%) 6 0.05 20.90064 0.1% GoX variant 20.72883 2.0 0.0502 7 0.025 20.11057 0.05% (dilution 6) 20.33254 2.0 0.0250 8 0.005 10.39779 0.025% (dilution 7) 40.43689 4.9 0.0051 9 0.003 30.29630 0.005% (dilution 8) 20.02033 1.7 0.0031 10 0.001 10.35204 0.003% (dilution 9) 20.06589 2.9 0.0010

[0274] Each of these samples was tested for the level of resting hydrogen peroxide using the Amplex Red hydrogen peroxide assay (TM-781, see Example 1), as well as set up for activation by addition of an equal mass of water and incubation at 37° C. for up to 24 hours.

[0275] The level of activated hydrogen peroxide was determined as described in Example 1.

TABLE-US-00006 TABLE 6 Resting [H.sub.2O.sub.2] μM 1 hour [H.sub.2O.sub.2] μM Ratio Sample 1 0.025% GOX 65.7 2425.38 369 Sample 1 0.005% GOX 21.9 11170.69 510 Sample 1 0.003% GOX 17.1 9506.756 557 Sample 1 0.001% GOX 10.6 4582.596 432 Sample 2 0.025% GOX 43.2 17097.48 395 Sample 2 0.005% GOX 26.9 9800.408 364 Sample 2 0.003% GOX 27.4 8444.577 308 Sample 2 0.001% GOX 27.8 2842.829 102

EXAMPLE 3

[0276]

TABLE-US-00007 TABLE 7 Glucose Ascorbic Glucose Fructose Water Glycerol Oxidase Acid Sample 1 5 78 9.9 7 0.1 0.5 (Variant 1) Sample 2 5 78 9.9 7 0.1 0.1 (Variant 2) All amounts % by weight

[0277] In order to prepare samples containing different levels of glucose oxidase, the samples were prepared by gravimetric mixing of a 0.1% and 0% glucose oxidase base preparations, with the masses being recorded to calculate exact dilutions used.

[0278] Each of the samples plus the supplied 0.1% GoX for each were then tested for the level of resting hydrogen peroxide and glucose oxidase activity using the Amplex Red hydrogen peroxide and glucose oxidase assays (TM-781 and TM-601; see above) respectively.

Resting and Stability Hydrogen Peroxide and Glucose Oxidase Levels

[0279] For glucose oxidase determination, approximately 0.1 g of each sample was weighed into 1.5 mL microfuge tubes and diluted with 900 μl PBS. For hydrogen peroxide, approximately 0.3 g of each sample was weighed into 1.5 ml microcentrifuge tubes and diluted with an equal mass of 99% ethanol. For both, the solutions were physically mixed using a sterile pipette tip prior to being centrifuged to remove precipitated material and then diluted to a suitable level using PBS. 50 μl of sample, standard curve point (hydrogen peroxide or glucose oxidase as required) or blank was pipetted into a 96 well plate and the reaction started by addition of 50 μl of the relevant substrate. For both assays, the absorbance at 570 nm was determined, for glucose oxidase, every 2 minutes for a total of 30 minutes and after 5 minutes for hydrogen peroxide. This was then used to produce the standard curve for each assay and quantify both the glucose oxidase activity and hydrogen peroxide level in each sample. The samples were also stored at 25° C. for 7 days and tested at 2, 5 and 7 days thereafter.

7 Day Stability Study of 0.25% Glucose Oxidase Formulation Containing Different Levels of Ascorbic Acid.

[0280]

TABLE-US-00008 TABLE 8 Time (days) Ascorbic acid % 0 2 5 7 0% ascorbic acid 476.6 1278.8 981.3 1618.8 0.5% ascorbic acid 50.1 61.6 59.7 69.1 [H.sub.2O.sub.2] μM

[0281] The results are illustrated in FIG. 6. The resting levels of hydrogen peroxide were reduced by the presence of ascorbic acid.

TABLE-US-00009 TABLE 9 Time (days) Ascorbic acid % 0 2 5 7 0% ascorbic acid 50474.7 16252.9 1190.1 2756.7 0.5% ascorbic acid 77285.3 64534.6 54056.1 34364.4 GOX activity (mU/ml)

[0282] The results are illustrated in FIG. 7. In the presence of ascorbic acid, the glucose oxidase has a higher initial activity and glucose oxidase activity is maintained at a higher level over time, over the testing period.

Simulated Wound Hydrogen Peroxide Level Determination

[0283] A simulated wound was established by weighing 0.5 g of the sample into a microfuge tube and centrifuging briefly to collect the gel at the bottom of the tube. On top of this was added 0.5 ml of horse serum and the tube incubated at 30° C. for up to 72 hours. During this time, 10 μl samples of the liquid supernatant (after mixing by inversion) were removed into either 140 μl of 99% ethanol for hydrogen peroxide measurement.

[0284] The hydrogen peroxide assay (TM-781) vortex mixed the sample briefly, followed by centrifugation at 12,000 rpm for 2 minutes to pellet precipitated material. The ethanol supernatant was further diluted with PBS and tested for hydrogen peroxide levels the Amplex Red assay kit.

72 Hour Hydrogen Peroxide Profiles

[0285]

TABLE-US-00010 TABLE 10 0.1% ascorbic acid 0.5% ascorbic acid Time Average [H202] Standard Average [H202] Standard (hours) (μM) deviation (μM) deviation 2 247 105.5 268.1 140 4 1777.8 801.3 573.6 16.1 8 2856.7 801.3 902.1 22.1 12 8364.2 457 851.2 127.3 24 6431 524.8 877.5 199.6 48 6754.2 326 1263.3 135.7 72 7568.8 46.4 1749.8 320.7 0.025% Glucose oxidase

[0286] The results are illustrated in FIG. 8.