ANTIMICROBIAL COMPOSITIONS AND FORMULATIONS

Abstract

Compositions for generating antimicrobial activity are described. The compositions comprise: a first phase; a second phase; an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme, wherein the first phase and the second phase are immiscible. The compositions may be formulated as emulsions or creams. Methods of making the compositions are described, as well as their use for the treatment of antimicrobial infections.

Claims

1. A composition for generating antimicrobial activity, comprising: a first phase; a second phase; an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme, wherein the first phase and the second phase are immiscible, and wherein the composition does not comprise sufficient free water to allow the enzyme to convert the substrate, wherein the composition comprises i) an inorganic salt and a viscosity-increasing agent and/or wherein ii) the composition comprises water that is additional to any water that may be present in the substance.

2. A composition according to claim 1 wherein the inorganic salt is magnesium sulfate heptahydrate.

3. A composition according to claim 1, wherein the first phase is less polar than the second phase, optionally wherein the first phase is a non-polar phase, and the second phase is a polar phase.

4. A composition according to any preceding claim, wherein the first phase is a lipophilic phase and the second phase is an aqueous phase.

5. A composition according to any preceding claim, wherein the second phase comprises a non-aqueous solvent, wherein the non-aqueous solvent is optionally glycerol, dimethylsulphoxide, propylene glycol or polyethylene glycol.

6. A composition according to claim 5, wherein the second phase comprises glycerol.

7. A composition according to any preceding claim, wherein the first phase comprises, an oil, wherein the oil is optionally olive oil, corn oil, vegetable oil, sunflower oil or paraffin oil.

8. A composition according to claim 7, wherein the first phase comprises paraffin oil.

9. A composition according to any preceding claim, comprising an emulsifier, preferably a surfactant, optionally wherein the surfactant is, or comprises, TWEEN, SPAN, Poloxamer or Polyglycerol polyricinoleate.

10. A composition according to claim 9, wherein the emulsifier is polyglycerol polyricinoleate.

11. A composition according to any preceding claim, which provides for sustained release of hydrogen peroxide at a level of less than 2 mmol/litre for a period of at least twenty four hours, following dilution of the composition.

12. A composition according to any preceding claim, which provides for sustained release of at least 0.1, 0.5, 1 or 1.5 mmol/litre hydrogen peroxide for a period of at least 24 hours.

13. A composition according to any preceding claim, wherein the enzyme is a purified enzyme.

14. A composition according to any preceding claim, wherein the enzyme is an oxidoreductase enzyme, preferably glucose oxidase.

15. A composition according to any preceding claim, wherein the substance lacks catalase activity and/or lacks peroxidase activity.

16. A composition according to any preceding claim, wherein the substance is an unrefined substance, such as an unrefined natural substance.

17. A composition according to any preceding claim, wherein the substance is a sugar substance, preferably comprising glucose.

18. A composition according to any preceding claim, wherein the substance is, or comprises, honey.

19. A composition according to any of claims 1 to 15, wherein the substance is, or comprises, a purified substrate for the enzyme, preferably a purified sugar such as purified glucose.

20. A composition according to claim 20, wherein the substance comprises a purified substrate for the enzyme and a purified solute in the form of a sugar or sugar derivative with a solubility of at least 100 g/100 g water at 20 C. and 1 atm, preferably greater than or equal to 200 g/100 g water at 20 C. and 1 atm, even more preferably greater than 300 g/100 g water at 20 C. and 1 atm.

21. A composition according to claim 20, wherein the substrate is glucose and the solute is fructose.

22. A composition according to any preceding claim, comprising 10 to 30% (v/v) non-aqueous solvent.

23. A composition according to any preceding claim, comprising 15 to 25% (v/v) non-aqueous solvent.

24. A composition according to any preceding claim comprising 30 to 50% (v/v) oil.

25. A composition according to any preceding claim comprising 35 to 45% (v/v) oil.

26. A composition according to any preceding claim, comprising 5 to 25% (v/v) emulsifier.

27. A composition according to any preceding claim, comprising 10 to 20% (v/v) emulsifier.

28. A composition according to any preceding claim, comprising 5 to 25% (v/v) of substance that comprises a substrate for the enzyme.

29. A composition according to any preceding claim, comprising 10 to 20% (v/v) of substance that comprises a substrate for the enzyme.

30. A composition according to any preceding claim, comprising 1 to 15% (v/v) additional water.

31. A composition according to any preceding claim, comprising 1 to 10% (v/v) additional water.

32. A composition according to any of claims 1 to 21, comprising 10-60%, preferably 20-50%, more preferably 35-45% (w/w) non-aqueous solvent.

33. A composition according to any of claims 1 to 21 or 32, comprising 10-40%, preferably 20-30% (w/w) oil.

34. A composition according to any of claims 1 to 21 or 32 to 33, comprising 1-10%, preferably 1-5% (w/w) emulsifier

35. A composition according to any of claims 1 to 21 or 32 to 34, comprising 10-50%, preferably, more preferably 20-40% (w/w) substance which comprises a substrate for the enzyme.

36. A composition according to any of claims 1 to 21, comprising 20-50% (w/w) non-aqueous solvent, 20-30% (w/w) oil, 1-5% (w/w) emulsifier and 20-40% (w/w) substance which comprises a substrate for the enzyme.

37. A composition according to any of claims 1 to 21, comprising 10-60% (w/w) non-aqueous solvent, 10-40% (w/w) oil, 1-10% (w/w) emulsifier and 10-50% (w/w) substance which comprises a substrate for the enzyme.

38. A composition according to any of claims 1 to 21, comprising 35-45% (w/w) non-aqueous solvent, 20-30% (w/w) oil, 1-5% (w/w) emulsifier and 25-35% (w/w) substance which comprises a substrate for the enzyme.

39. A composition according to any preceding claim, in which the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance.

40. A composition according to any preceding claim, which does not include any detectable hydrogen peroxide or comprises substantially no hydrogen peroxide.

41. A composition according to any preceding claim, wherein the composition is a sterile composition.

42. A composition according to any preceding claim, which comprises greater than 20% (w/w) water and less than or equal to 40% (w/w) water, preferably less than or equal to 30% (w/w) water.

43. A composition according to any preceding claim which contains essentially no zinc oxide or which comprises substantially no zinc oxide.

44. A method of making a composition, comprising: mixing a first component; a second component; an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme to form the composition, wherein the first component and second component are immiscible and optionally mixing i) a viscosity increasing agent and an organic salt and/or ii) water that is additional to any water that may be present in the substance.

45. A method according to claim 44, wherein the first component comprises an oil and the second component comprises a non-aqueous solvent.

46. A method according to claim 44 or claim 45, comprising mixing an emulsifier.

47. A method according to claim 46, comprising the steps of: a) adding the water to the second component; b) mixing the second component and the additional water; c) adding the enzyme and the substance to the mixture from step b); d) mixing the enzyme and the substance with the mixture of step b); e) adding the emulsifier to the mixture from step d); f) mixing the emulsifier with the mixture from step d).

48. A method according to claim 47, wherein steps b), d) and f) are carried out at a shear rate of 1000 1/s to 4000 1/s.

49. A method according to claim 48, wherein the shear rate is 2500 1/s to 3500 1/s.

50. A method according to any of claims 47 to 49, wherein steps b), d) and f) are carried out at 35 to 45 C., preferably about 40 C.

51. A method according to any of claims 47 to 50, wherein the mixing in steps b) and d) is carried out for at least a minute, preferably at least 2 minutes.

52. A method according to any of claims 47 to 51, wherein the mixing in step f) is carried out for at least 5 minutes, preferably at least 10 minutes.

53. A method according to claim 46, wherein the emulsifier is pre-mixed with the first component.

54. A method of making a composition, comprising: mixing a first component; a second component; an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme to form the composition, wherein the first component and second component are immiscible and wherein the emulsifier is pre-mixed with the first component.

55. A method according to claim 53 or claim 54, wherein the pre-mixing occurs using a homogeniser at 5,000 rpm or greater, preferably 7,500 rpm or greater, preferably 10,000 rpm or greater.

56. A method according to any of claims 53 55, wherein the pre-mixing occurs for at least 30 seconds, preferably at least one minute, preferably at least 2 minutes.

57. A method according to any of claims 53 to 56, wherein following pre-mixing, the second component, the enzyme and the substance comprising the substrate for the enzyme are added, preferably at a rate of less than 5 ml per minute, more preferably less than 3 ml per minute, most preferably less than 2 ml per minute.

58. A method according to claim 57, wherein once the second component, the enzyme and the substance comprising the substrate for the enzyme have been added to the first phase, homogenisation takes place for at least 5 minutes, preferably at least 10 minutes.

59. A method according to claim 58, wherein homogenisation takes place at 5,000 rpm or greater, preferably 7,500 rpm or greater, preferably 10,000 rpm or greater.

60. A composition comprising an oil, an emulsifier, an enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme, wherein: a) the substance and enzyme has been mixed with the oil in a ratio of 30:70 to 70:30 (by volume); and/or b) the composition comprises 1-5% (by volume) of emulsifier, optionally 1.5 to 3% (by volume).

61. A composition according to claim 60, wherein the substance and enzyme has been mixed with the oil in a ratio of at least 50:50 (by volume). for example 60:40 (by volume).

62. A composition according to claim 60 or claim 61, wherein the oil is paraffin oil.

63. A composition according to any of claims 60 to 62, wherein the emulsifier is PGPR.

64. A composition according to any of claims 60 to 63, wherein the substance is a sugar substance.

65. A composition according to claim 64, wherein the sugar substance comprises glucose and fructose.

66. A method of forming a composition comprising mixing an oil, an emulsifier, an enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme, wherein the substance and enzyme has been mixed with the oil in a ratio of 30:70 to 70:30 (by volume), and/or wherein emulsifier is added in an amount of 1-5% (by volume).

67. A method according to claim 66, comprising mixing the substance and enzyme with the oil in a ratio of at least 50:50 (by volume). for example 60:40 (by volume).

68. A composition according to any of claims 1 to 43, for use as a medicament.

69. A composition according to any of claims 1 to 43, for use in the prevention or treatment of a microbial infection.

70. Use of a composition according to any of claims 1 to 43, in the manufacture of a medicament for the prevention or treatment of microbial infection.

71. A method of preventing or treating a microbial infection which comprises administering an effective amount of a composition according to any of claims 1 to 43, to a subject in need of such a treatment.

72. A spraying device comprising a composition as defined in any of claims 1 to 43.

Description

[0328] Embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

[0329] FIG. 1 shows the results of an assay for the cytotoxic activity of Surgihoney;

[0330] FIG. 2A shows different hydrogen peroxide production rates for Surgihoney SH1, SH2, and SH3;

[0331] FIG. 2B shows the relationship between phenol activity and maximum hydrogen peroxide activity in Surgihoney SH1, SH2, and SH3;

[0332] FIG. 3 shows time kill curves for Surgihoney 1 (S1), Surgihoney 3 (S3), and Medihoney (MH) for different test organisms: (a) Staphylococcus aureus; (b) Methicillin-resistant Staphylococcus aureus (MRSA); (c) E. coli; (d) vancomycin resistant enterococcus (VRE); (e) Pseudomonas aeruginosa; (f) Klebsiella; (g) E. coli ESBL; (h) Enterococcus faecalis;

[0333] FIG. 4 shows an optical microscopy images of reverse micelles in an emulsion containing Surgihoney;

[0334] FIG. 5 shows the stability of an emulsion containing Surgihoney over a time period of one month;

[0335] FIG. 6 shows reverse micelles formed in an emulsion containing Surgihoney;

[0336] FIG. 7 shows the spray produced by an emulsion containing Surgihoney;

[0337] FIG. 8 shows the stability of emulsions containing Surgihoney with additional water in comparison to a control emulsion containing Surgihoney, but without the addition of water;

[0338] FIG. 9 shows pCT images of an emulsion containing Surgihoney which shows particle/micellular distribution and size within the emulsion;

[0339] FIG. 10 shows optical microscope images of particle/micellular distribution and size within an emulsion containing Surgihoney;

[0340] FIG. 11 shows viscosity of emulsions containing Surgihoney with additional water in comparison with emulsions containing Surgihoney without additional water at a shear rates of 100 1/s and 1000 1/s;

[0341] FIG. 12 shows the viscosity-temperature relationship between a formulation containing Surgihoney without addition of water and an emulsions containing Surgihoney with additional water;

[0342] FIG. 13 shows concentrations of hydrogen peroxide produced by an emulsion containing Surgihoney, compared to Surgihoney, assuming a Surgihoney concentration of 1 g/100 ml;

[0343] FIG. 14 is a graph showing the effect of compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to Surgihoney, at various concentrations;

[0344] FIG. 15 is a graph showing the effect of sterile and non-sterile compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) (buffered at pH 4.03) on the growth of planktonic MRSA, at various concentrations;

[0345] FIG. 16 is a graph showing the effect of sterile and non-sterile compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) (unbuffered) on the growth of planktonic MRSA, at various concentrations;

[0346] FIG. 17 is a graph showing the effect of sterile and non-sterile compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) (buffered at pH 7.04) on the growth of planktonic MRSA, at various concentrations;

[0347] FIG. 18 is a table showing the effect of sterile and non-sterile compositions for use in the invention comprising glucose, glucose oxidase and fructose, on the MIC and MBC of planktonic MRSA, at various concentrations;

[0348] FIG. 19 shows the effect of compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

[0349] FIG. 20 shows the effect of SyntheticRO on the MIC and MBC of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

[0350] FIG. 21 shows the stability of emulsions containing different amounts of SurgihoneyRO and PGPR over a 7 day period.

[0351] FIG. 22 shows the viscosity at varying shear rates of emulsions containing different amounts of SurgihoneyRO after a 7 day period;

[0352] FIG. 23 shows G and G measurements of emulsions containing different amounts of SurgihoneyRO after 7 days; and

[0353] FIG. 24 shows rhe characterisation of the SurgihoneyRO 60:40 (2% PGPR) emulsion showing the relative viscosity (A), the effect of frequency on G and G (B), the size of the reverse micelles within the emulsion (C) and the rate of hydrogen peroxide release from the final product (D).

[0354] Surgihoney may also be referred to as Surgihoney, SurgihoneyRO, SurgihoneyRO or SHRO. Compositions of the invention (or for use in the invention), such as compositions which comprise purified glucose, purified fructose and glucose oxidase may be referred to as SyntheticRO, synthetic honey compositions or synthetic compositions.

EXAMPLE 1

Surgihoney Cream Formulation

[0355] A cream formulation comprising Surgihoney SH1 was made with the following ingredients: [0356] beeswax (for the lipophilic phase); [0357] soya lecithin (as an emulsifier); [0358] water; and [0359] SH1 Surgihoney.

[0360] The cream remains stable for many years, as judged by its ability to produce hydrogen peroxide when contacted with water.

EXAMPLE 2

Anti-Viral Activity of Surgihoney

[0361] SH1 or SH2 Surgihoney was mixed with Herpes Simplex Virus Type 1 or 2 (HSV 1 or HSV 2) in cell culture medium (a 50% mixture of honey and virus in cell culture medium) and then incubated for 1 hour at 37 C. A dilution series was then made from the mixture, and the dilutions were plated onto Vero cells. SH1 Surgihoney reduced the titre of virus by 1 log. SH2 Surgihoney was virucidal for both HSV 1 and HSV 2 (>6 log drop in titre). The experiment was repeatable.

EXAMPLE 3

Anti-Viral Activity of Surgihoney

[0362] SH1 or SH2 Surgihoney was mixed with Herpes Simplex Virus (HSV) (50 g honey and 50 l virus) and incubated for 1 hour at 37 C. A dilution series (10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5) was then made from the mixture, and the dilutions were used in a plaque reduction assay. Controls with no honey, or with control honey were also performed. The number of viral plaques formed for each dilution was recorded. The results are shown in the Table below.

TABLE-US-00006 TABLE 2 Anti-viral effect of Surgihoney Experiment 2 Experiment 1 well Honey Dilution well 1 well 2 well 3 well 1 well 2 3 SH1 2 * * * * * * 3 1 1 5 0 0 0 4 0 1 1 0 0 0 5 0 0 0 0 0 0 SH2 2 * * * * * * 3 0 0 0 0 0 0 4 0 0 0 0 0 0 5 0 0 0 0 0 0 Control 2 100 95 88 108 128 106 Honey 3 13 15 11 14 12 15 4 2 1 2 3 2 2 5 0 0 0 0 0 1 No 2 160 158 164 Honey 3 28 22 18 4 6 4 1 5 1 0 1

[0363] The results show that SH1 and SH2 Surgihoney was strongly virucidal against HSV in both experiments.

EXAMPLE 4

Cytotoxic Activity of Surgihoney

[0364] SH1 or SH2 Surgihoney (50 g honey diluted 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5) was incubated on cells for 2 days. The number of live cells, and the total number of cells was counted (percentage viability=live/total100). The results are shown in the table below, and in FIG. 1.

[0365] The results show that SH1 Surgihoney was cytotoxic at the 10.sup.2 dilution, and cytostatic at the 10.sup.3 and 10.sup.4 dilutions, and that SH2 Surgihoney was cytostatic at the 10.sup.2, 10.sup.3 and 10.sup.4 dilutions. SH1 and SH2 Surgihoney were not cytotoxic or cytostatic at the 10.sup.5 dilution.

[0366] It is concluded from the results in Examples 3 and 4 that Surgihoney can be administered at doses which are virucidal but not cytotoxic or cytostatic.

TABLE-US-00007 TABLE 3 Cytotoxic activity of Surgihoney Number of live cells Total number of cells Percentage viability Con- standard standard standard dition Dilution rep1 rep2 Ave deviation rep1 rep2 Ave deviation rep1 rep2 Ave deviation DMEM 1300000 2100000 1700000 565685.4 1400000 2600000 2000000 848528.1 92.9 80.8 86.8 8.5 Control 2 1200000 880000 1040000 226274.2 1300000 1000000 1150000 212132 92.3 88.0 90.2 3.0 honey 3 2700000 2400000 2550000 212132 2800000 2600000 2700000 141421.4 96.4 92.3 94.4 2.9 4 3400000 2800000 3100000 424264.1 3600000 3000000 3300000 424264.1 94.4 93.3 93.9 0.8 5 2100000 1300000 1700000 565685.4 2200000 1500000 1850000 494974.7 95.5 86.7 91.1 6.2 SH1 2 120000 70000 95000 35355.34 370000 350000 360000 14142.14 32.4 20.0 26.2 8.8 3 380000 380000 380000 0 400000 600000 500000 141421.4 95.0 63.3 79.2 22.4 4 430000 780000 605000 247487.4 470000 850000 660000 268700.6 91.5 91.8 91.6 0.2 5 1800000 2200000 2000000 282842.7 2000000 2400000 2200000 282842.7 90.0 91.7 90.8 1.2 SH2 2 320000 360000 340000 28284.27 390000 400000 395000 7071.068 82.1 90.0 86.0 5.6 3 450000 570000 510000 84852.81 760000 730000 745000 21213.2 59.2 78.1 68.6 13.3 4 460000 690000 575000 162634.6 660000 790000 725000 91923.88 69.7 87.3 78.5 12.5 5 1600000 1700000 1650000 70710.68 1800000 2000000 1900000 141421.4 88.9 85.0 86.9 2.7

EXAMPLE 5

Antimicrobial Activity of Surgihoney

[0367] The antimicrobial activity of Surgihoney (SH) and two prototype modified honeys made by Apis mellifera (honeybee) against Staphylococcus aureus (NCIMB 9518) was tested. We also examined a number of modified types of Surgihoney for the ability to change the level of production of hydrogen peroxide from the samples.

[0368] Methods: Surgihoney (SH) was compared with two modified honeys, Prototype 1 (PT1) and Prototype 2 (PT2) using a bioassay method against a standard strain of Staphylococcus aureus. Further work studied the rate of generation of hydrogen peroxide from these preparations.

[0369] Results: Surgihoney antimicrobial activity was shown to be largely due to hydrogen peroxide production. By modification of Surgihoney, two more potent honey prototypes were shown to generate between a two- and three-fold greater antibacterial activity and up to ten times greater peroxide activity.

[0370] Conclusions: Surgihoney shows good antimicrobial activity. Two further honey prototypes have been shown to have antimicrobial activity that is possible to be enhanced due to demonstrated increases in peroxide activity.

Methods

1. Determination of Honey Activity by Bioassay Method

[0371] The antibacterial activity of Surgihoney (S) and two modified honeys, Prototype 1 (PT1) and Prototype 2 (PT2) was measured using Staphylococcus aureus (NCIMB 9518) and expressed as the equivalent percentage phenol. Values were calculated of the mean from three sample replicates tested, repeated on three days.

[0372] Assay Method. The agar well diffusion method used was adapted from the punch plate assay for inhibitory substances described in the Microbiology Standard Methods Manual for the New Zealand Dairy Industry (1982) [Bee Products Standards Council: Proposed standard for measuring the non-peroxide activity of honey. In. New Zealand: Bee Products Standards Council; 1982.].

[0373] Inoculum Preparation. Overnight culture was adjusted to an absorbance of 0.5 measured at 540 nm using sterile nutrient broth as a blank and a diluents and a cuvette with a 1 cm pathway.

[0374] Assay Plate preparation. A volume of 100 l of the culture adjusted to 0.5 absorbance was used to seed 150 ml nutrient agar to make the assay plates. The agar was swirled to mix thoroughly and poured into large petri dishes which had been placed on a level surface. As soon as the agar was set the plates were placed upside down overnight before using the next day. For assay these seeded plates were removed from 4 C. and allowed to stand at room temperature for 15 min before cutting 7.0 mm diameter wells into the surface of the agar. 250 l of test material (sample or standard) was placed into each well.

[0375] Catalase solution. A 200 mg/ml solution of catalase from bovine liver (Sigma C9322, 2900 units/mg) in distilled water was prepared fresh each day.

[0376] Sample preparation. Primary sample solutions were prepared by adding 4 g of sample to 4 ml of distilled water in universals and placed at 37 C. for 30 minutes to aid mixing. To prepare secondary solutions, 2 ml of the primary sample solution was added to 2 ml of distilled water in universals and mixed for total activity testing and 2 ml of the primary sample solution was added to 2 ml of catalase solution and mixed for non-peroxide activity.

[0377] Preparation of phenol standards. Standards (w/v) 10%, 30%, 50% phenol were prepared by dissolving phenol in water. Phenol standards were brought to room temperature in the dark before use and were mixed thoroughly before addition to test wells. Each standard was placed in three wells to test in triplicate. Standards were kept at 4 C. with an expiry date of one month.

[0378] Sample and standard application. All samples and standards were tested in triplicate by adding 250 l to each of 3 wells.

[0379] Plate incubation. After application of samples the plates were incubated for approximately 18 hours at 37 C. The diameter of inhibition zones, including the diameter of the well (7.0 mm), was recorded.

[0380] Calculation of antibacterial activity of samples. The mean diameter of the clear zone around each phenol standard was calculated and squared. A standard graph was plotted of % phenol against the square of the mean diameter of the clear zone. A best-fit straight line was obtained using linear regression and the equation of this line was used to calculate the activity of each diluted honey sample from the square of the mean measurement of the diameter of the clear zone. To allow for the dilution (assuming the density of the Surgihoney to be 1.35 g/ml) this figure was multiplied by a factor of 4.69 and the activity of the samples was then expressed as the equivalent phenol concentration (% w/v).

[0381] Total Activity: all the activity, including activity due to hydrogen peroxide (H.sub.2O.sub.2).

[0382] Non-Peroxide Activity: H.sub.2O.sub.2 is removed by treating samples with catalase enzyme.

2. Determination of Honey Activity by H.sub.2O.sub.2 Method

[0383] The activity was measured using the Merckoquant 1.10011. & 1.10081.

[0384] Peroxide Test Kits. Concentrations expressed as the equivalent mg/L H.sub.2O.sub.2.

[0385] Samples were diluted 1:10 with purified water. Following 5 min incubation, all samples were measured for H.sub.2O.sub.2 production each hour over a 12 hour period followed by 24 and 48 hour time points.

[0386] Method of Determination. Peroxidase transfers oxygen from the peroxide to an organic redox indicator, which is then converted to a blue coloured oxidation product. The peroxide concentration is measured semi-quantitatively by visual comparison of the reaction zone of the test strip with the fields of a colour scale. The reaction zone of the test strip is immersed into the Surgihoney sample for 1 sec, allowing excess liquid to run off the strip onto an absorbent paper towel and after 15 seconds (Cat. No. 110011), 5 seconds (Cat. No. 110081), after which a determination of the colour formed in the reaction zone more precisely coincided with the colour fields scale.

Results

1. Activity Rating

[0387] The antimicrobial activity produced by the modification of the honey samples resulted in a two-fold and almost three-fold respectively increase in phenol activity with PT1 and PT2 compared with Surgihoney alone. The results for the three samples of Surgihoney (SH) and two modified prototypes, PT1 and PT2 are shown in the Table below.

TABLE-US-00008 TABLE 4 The peroxide and non-peroxide antibacterial activities of Surgihoney (SH) and two modified prototypes, PT1 and PT2 against Staphylococcus aureus (NCIMB 9518). Total Non-Peroxide Activity Activity Sample Name Batch No. (% phenol) (% phenol) Surgihoney 2015-06-018B 32 0 Surgihoney PT1 HHI4110311 65 7 Surgihoney PT2 HHI14110312 83 10
2. Determination of Honey Activity by H.sub.2O.sub.2 Method

[0388] The prototype modifications are observed to generate up to seven and ten times the hydrogen peroxide activity of Surgihoney. The results for the three samples are shown in FIG. 2A. By taking the maximum level of hydrogen peroxide output for each of the three honey prototypes and plotting this against the total phenol activity a linear relationship is observed (FIG. 2B).

Discussion

[0389] The results from this work show that the main antimicrobial activity of Surgihoney and two modified prototypes, PT1 and PT2 are due to hydrogen peroxide. This is a similar finding to certain other honeys from a variety of floral sources. However, unlike previous work the availability of hydrogen peroxide from the samples is able to be enhanced and at 12 hours is seven and ten times respectively the value for Surgihoney alone. There is a striking linear relationship between the antimicrobial activity and the maximum output of hydrogen peroxide from the three honey prototypes.

[0390] This peroxide activity offers potent antimicrobial activity that is ideally suited to treat or prevent microbial infections. Hydrogen peroxide is an effective antimicrobial and is already used as a biocide for its potent activity against vegetative bacteria, yeasts and spores. It produces its antimicrobial effect through chemical oxidation of cellular components.

[0391] The human toxicity of hydrogen peroxide is concentration dependent and one study has claimed that the differential concentrations for antimicrobial and human toxicity might overlap. By contrast, certain preparations of honey have been shown to be an effective antimicrobial agent by supplying low concentrations of hydrogen peroxide continuously over time rather than as a large amount and without such toxicity. Indeed there is compelling evidence that where physiological levels of hydrogen peroxide are applied to mammalian cells there is a stimulation of biological responses and activation of specific biochemical pathways in these cells.

[0392] Clearly Surgihoney and the two modified prototypes, PT1 and PT2 offer effective hydrogen peroxide release over at least 24 hours.

Conclusions

[0393] Surgihoney and the two modified prototypes, PT1 and PT2 have been shown to have potent antimicrobial activity against a standard strain of Staphylococcus aureus. These antimicrobial activities have been shown to be due to hydrogen peroxide. The activity is scalable and can be described in terms of hydrogen peroxide activity. These modified honeys are effective, non-toxic and easy to administer.

EXAMPLE 6

In Vitro Antimicrobial Activity of Surgihoney

[0394] This example describes susceptibility testing of a range of bacterial isolates to Surgihoney by disc diffusion method, minimum inhibitory concentration (MIC) and minimum cidal concentration (MBC) determination, and time bactericidal measurements.

SUMMARY

[0395] Results: Surgihoney demonstrates highly potent inhibitory and cidal activity against a wide range of Gram positive and Gram negative bacteria and fungi. MIC/MBC's are significantly lower than concentrations likely to be achieved in topical clinical use. Surgihoney 1 MIC/MBC's for Staph. Aureus are 31 and 125 gms/L and Surgihoney 3 MIC/MBC's 0.12 and 0.24 gms/L. Cidal speed depends on the potency. In Surgihoney 1, the least potent, complete cidal activity occurs for all organisms tested within 48 hours. For Surgihoney 3, the most potent, cidal activity occurs within 30 minutes. Maintenance of the Surgihoney inoculums preparation for up to a week demonstrated complete cidal activity and no bacterial persistence.

[0396] Conclusions: Surgihoney has wide potential as a highly active topical treatment combining the effects of the healing properties of honey with the potent antimicrobial activity of the bioengineered product. It is highly active against multidrug resistant bacteria. It is more active than other honeys tested and comparable to chemical antiseptics in antimicrobial activity.

[0397] This study examines the in-vitro properties of Surgihoney. Surgihoney retains all the established healing properties of natural honey but its antimicrobial activity can be set at whichever potency is required. This study determined minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of Surgihoney 1, 2 and 3 and time kill curves.

Methods

[0398] Surgihoney was provided as potency grades 1, 2 and 3. It was presented as a sterile pharmaceutical grade product in a sachet in semisolid form.

[0399] Clinical isolates were collected from soft tissue microbiology samples. Eighteen isolates of Staphylococcus aureus, 12 methicillin-sensitive (MSSA) and 6 methicillin-resistant (MRSA), 6 isolates of haemolytic streptococci, Lancefield groups A (2), B (2), C (1), G (1), 5 isolates of Enterococcus spp. Including vancomycin-resistant E. faecium, 6 of Esch. coli, including extended spectrum lactamase producers, 2 of Klebsiella spp., 1 Serratia Marcescens Amp C producer, 4 of Pseudomonas aeruginosa, 1 of Acinetobacter lwoffii, 1 of Propionibacterium acnes, 1 Bacteroides fragilis, and 2 of Candida albicans, 1 of Candida glabrata, 1 of Aspergillus fumigates were tested against Surgihoney.

Agar Diffusion

[0400] Six mm wells were cut in isosenitest agar which had already been inoculated with the test organism at a concentration to give a semiconfluent growth. Test Surgihoney and other honeys in the pilot study were added to the wells.

[0401] A pilot study was carried out initially to compare Surgihoney potencies 51, S2, S3 with a variety of honeys from around the world, European, South American, New Zealand, Yemani, Sudanese and with medical honey, Medihoney and with antimicrobial dressings containing silver (Silver Aquacell) and iodine (Iodoflex). Wells were cut in the plates inoculated with Staphylococcus aureus and filled with test honey or in the case of the dressings, these were cut to 22 cm and placed on the surface of the inoculated plates.

[0402] Following the pilot studies the Surgihoney potencies 51, S2, S3 were tested alone against the range of bacterial isolates from skin lesions. The wells were filled to the surface with a preparation of approximately 2 gms neat Surgihoney of the three potencies, diluted and emulsified in an equal volume of sterile water. Zone sizes were measured after 18-24 hours aerobic incubation (longer for Candida and Aspergillus spp., and anaerobically for Propionibacterium sp. And Bacteroides sp.)

Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations

[0403] Surgihoney product was warmed to 37 C. to liquefy it and 5 gms was mixed with 10 mL sterile deionised water. This dilution was regarded as the neat substance for serial dilution. The British Society of Antimicrobial Chemotherapy (BSAC) method for performing minimum inhibitory concentrations (MIC's) and minimum bactericidal concentrations (MBC's) was used (Andrews J M. Determination of minimum inhibitory concentrations. J Antimicrob 372 Chemother 2001; 48(Supp 1): 5-16). The Surgihoney products were serially diluted in microtitre tray wells from neat to 1 in 1024. 75 L of each honey dilution was added to each well in the strip of the microtitre tray. The neat concentration represented a concentration of 250 gm/L and the 1 in 2048 dilution, approximately 0.12 gm/L.

[0404] The test organisms were prepared by taking four morphologically identical colonies for each organism from pure culture to create a 0.5 McFarland density. This was further diluted 1:10.

[0405] All wells including controls were inoculated with 75 L of the test isolate preparation. The well trays were incubated at 37 C. for 18 hours. The MIC was regarded as the most dilute well that showed no detectable turbidity.

[0406] The MIC well and those around the MIC well were sub-cultured on blood agar and incubated at 37 C. for 18 hours to determine the MBC. The MBC was the most dilute concentration which showed no growth after incubation.

Time Kill Curves

[0407] The test organism inoculums was prepared by taking 0.1 mL of a 0.5 MacFarlane density of the test organism and inoculating this in 3 mL of nutrient broth. The test inoculums was divided into 3 separate bijous, a control and three test preparations to which were added 0.5 g of Surgihoney 1 (S1), Surgihoney 3 (S3) or Medihoney (MH). Colony counts of the inocula were determined by serial dilution 1:10 and plating 0.1 mL on a blood agar plate, repeated 3 times.

[0408] The test and control inocula were kept at 30 C. to simulate the temperature of a superficial skin lesion. Colony counts were performed as above in triplicate at time 0.5, 2, 4, 24, 48, 72 and 168 hours.

[0409] A terminal culture was performed by inoculating 0.1 ml of the original inoculums into nutrient broth to neutralise any residual effect of the Surgihoney and incubating for 72 hours at 37 C., before plating on blood agar to determine test organism survival.

Results

Inhibitory Zone Sizes.

[0410] The pilot comparative studies demonstrated that all the Surgihoney potencies had greater antimicrobial activity than any other honey tested including the medical grade honey, Medihoney. The inhibitory zones for S1 were larger than those produced by any other honey. Silver dressings produced some inhibitory effect beneath the dressing but there was no zone of inhibition as there was for Surgihoney. Iodine dressings produced a large zone of inhibition (approximately 70 mm) to Staphylococcus aureus, larger than S1 (36 mm) and equivalent to S3 (67 mm).

[0411] In the quantitative zone size testing, Surgihoney at all potencies produced an inhibitory zone in agar diffusion against all bacteria tested, both Gram positive and Gram negative bacteria including multiply antibiotic resistant bacteria, and fungal species. The zone size for each species increased with increasing Surgihoney potency preparations. Table 5. The inhibitory effect of Surgihoney was not dependant only on direct contact with the active agent as with the silver dressings, but diffused well beyond the well producing the extensive zones listed in Table 5.

MIC's & MBC's

[0412] Surgihoney demonstrated significant antimicrobial activity against all the isolates tested. MIC's and MBC's were very consistent amongst isolates of the same species whether the isolates were multidrug resistant or highly sensitive. Table 6 lists the MIC and MBC values for isolate species tested by dilution ratio and Table 7 shows the MIC and MBC's in grams per litre. The degree of potency rose with the grade of Surgihoney. The MBC for each isolate was close to the MIC within a single dilution in most cases.

[0413] Topical concentration of Surgihoney is estimated at approximately 500 gms/L. Surgihoney 1 MIC/MBC's for Staph. Aureus are 31 and 125 gms/L and Surgihoney 3 MIC/MBC's 0.12 and 0.24 gms/L respectively.

Time Kill Curves.

[0414] Surgihoney kills bacteria rapidly. Starting with a colony forming units per millilitre (cfu/mL) of approximately 105, cfu/mL numbers in the control rose steadily, whereas in the Surgihoney inocula the cfu/mL fell rapidly after contact with both potencies of Surgihoney. By 30 minutes cfu numbers had fallen 1000 fold in most cases for both S1 and S3 (FIG. 3). For S1 bacterial growth was undetectable by 2 hours in most cases and for S3 by 30 minutes. Enterococci appeared more resilient and persisted for 48 hours. Cidal activity was complete for all organisms as terminal culture in nutrient broth with subsequent plating on blood agar failed to detect any organism in the S1 or S3 inocula.

Discussion

[0415] Surgihoney is natural honey which is also organic in the current sense of the word in that it has no agricultural additives or antimicrobial residues unlike much commercial honey for human consumption. It is not dependant on particular nectar sources, unlike honeys such as manuka which depends on a specific plant nectar source for its enhanced activity. The antimicrobial activity can be controlled in Surgihoney by the preparation process allowing the production of different grades with measured potency which is consistent.

[0416] This study has clearly demonstrated the efficacy of Surgihoney as a highly potent antimicrobial, active against all species of bacteria and fungi tested. In the preliminary pilot studies comparing Surgihoney with a variety of honeys sourced from around the world and with medical grade honey, Medihoney, Surgihoney demonstrated significantly greater antimicrobial efficacy. By comparison with the commonly used topical antiseptics silver and iodine, Surgihoney 3 produced an antimicrobial effect as great as iodine dressings and greater than silver dressings (Aquacel Ag) which was only effective at inhibiting bacteria in direct contact with the dressing.

[0417] MIC and MBC testing show that Surgihoney not only inhibits but also kills microbes at concentrations 10 to a 1000 fold below those that are likely to be achieved in topical treatment, estimated at 500 gms/L. The cidal activity of Surgihoney occurs at concentrations close to its inhibitory activity. There is therefore the potential for Surgihoney to be highly active in polymicrobial inhibition and eradication when applied topically.

[0418] The speed of cidal activity is shown by the time kill curves to be extremely rapid, within 30 minutes for Surgihoney 3 and within 2 hours for Surgihoney 1. This is the case for both Gram-positive and Gram-negative organisms, although enterococci appear slightly more resilient. Fungi, Candida spp. Aspergillus sp. also require higher concentrations and more prolonged exposure to inhibit growth and kill the organism.

[0419] These in vitro studies have demonstrated the potential of Surgihoney with high antimicrobial activity whose potency can be controlled.

Conclusion

[0420] These in vitro results support the clinical use of Surgihoney as a potent and non-toxic antimicrobial.

TABLE-US-00009 TABLE 5 Inhibitory zones sizes with different potencies of Surgihoney (S1, S2, S3) No. of S1 Mean zone S2 Mean zone S3 Mean zone Bacteria strains (range)/mm (range)/mm (range)/mm Methicillin-sensitive 12 36.2 (32-38) 53.4 (44-58) 66.5 (60-72) Staphylococcus aureus (MSSA) Methicillin-resistant 6 35.6 (31-38) 52.6 (48-59) 67.3 (59-73) Staphylococcus aureus (MRSA) Streptococci Beta 6 40.0 (35-42) 44.5 (38-51) 59.2 (53-69) haemolytic Enterococcus spp 5 38.0 (34-39) 49.5 (44-55) 61.8 (59-64) Escherichia coli 6 33.4 (30-37) 49.5 (36-55) 62.7 (59-69) Klebsiella sp. 2 34.2 (30-38) 40.0 (38-42) 57.0 (52-62) Pseudomonas 4 25.8 (20-28) 34.8 (30-38) 50.2 (46-51) aeruginosa Acinetobacter lwoffii 1 32.1 43.7 55.2 Bacteroides fragilis 1 22.3 28.7 34.2 Propionibacterium 1 19.7 23.4 31.9 acnes Candida sp. 2 9 (8-10) 15 (15) 26 (24-28) Aspergillus 1 8 12 18 fumigatus

TABLE-US-00010 TABLE 6 Serial double dilutions from neat Surgihoney (S1, S2, S3) showing dilution of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). S1 S2 S3 Organism name MIC MBC MIC MBC MIC MBC MSSA 1:8 1:2 1:32 1:16 1:2048 1:1024 MRSA 1:16 1:4 1:32 1:16 1:2048 1:1024 Group B 1:64 1:16 1:64 1:64 1:1024 1:256 Streptococci Group A 1:32 1:16 1:128 1:64 1:1024 1:512 Streptococci Enterococcus 1:8 1:2 1:32 1:4 1:256 1:64 E. coli 1:8 1:4 1:64 1:64 1:256 1:128 E. coli ESBL 1:8 1:2 1:64 1:64 1:256 1:128 Serr. liquefaciens 1:8 1:4 1:16 1:4 1:256 1:128 Amp C Kleb. pneumoniae 1:4 1:2 1:32 1:32 1:256 1:128 Pseud. aeruginosa 1:16 1:16 1:64 1:16 1:256 1:64 Candida albicans Turbid at Growth at 1:16 1:16 1:64 1:64 neat neat

TABLE-US-00011 TABLE 7 Surgihoney MIC and MBC values expressed in Grams/Litre S1 S2 S3 Organism name MIC MBC MIC MBC MIC MBC MSSA 31 125 7.8 15.6 0.12 0.24 MRSA 15.6 62.5 7.8 15.6 0.12 0.24 Group B 3.9 15.6 3.9 3.9 0.24 0.9 Streptococci Group A 7.8 15.6 1.9 3.9 0.24 0.48 Streptococci Enterococcus 31 125 7.8 62.5 0.9 3.9 E. coli 31 62.5 3.9 3.9 0.9 1.9 E. coli ESBL 31 125 3.9 3.9 0.9 1.9 Serr. liquefaciens 31 62.5 15.6 62.5 0.9 1.9 Amp C Kleb. pneumoniae 1:4 125 7.8 7.8 0.9 1.9 Pseud. aeruginosa 15.6 15.6 3.9 15.6 0.9 3.9 Candida albicans Turbid at Growth at 15.6 15.6 3.9 3.9 neat neat

EXAMPLE 7

Surgihoney Emulsion

Preparation

[0421] 10 g Surgihoney was dissolved in 10 ml of glycerol. 10 ml of paraffin oil was then added to a Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 1 ml of PGPR (Polyglycerol polyricinoleate) was then added. The rheometer was then started under the following conditions; Shear rate 2000 1/s, Temperature set at 37.5 C. After 2 minutes, 10 ml of Surgihoney-glycerol solution was added dropwise. Once a total of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier to a container.

Optical Microscopy

[0422] Optical microscopy revealed that the emulsion contained reverse micelles which encapsulated Surgihoney. Such micelles can be observed in FIG. 4. The average micelle diameter was found to be 178 m.

Hydrogen Peroxide Tests

[0423] Hydrogen peroxide stick tests (Purchased from Sigma Aldrich (Quantofix)) were used to detect hydrogen peroxide in the emulsion. The tests were carried out before and after addition of water, and showed that before addition of water, the emulsion produced no hydrogen peroxide, and after water was added, the emulsion tested positive for hydrogen peroxide. A positive test was indicated by a colour change to blue.

Stability Test The emulsion maintained its capacity to generate hydrogen peroxide after storage at ambient conditions for at least four weeks.

Spray Test

[0424] The emulsion was added to a pump-action spray bottle and was found to be sprayable.

EXAMPLE 8

Effects of Different Parameters on Stability of Surgihoney Emulsions

[0425] The effects of changing the Surgihoney emulsion preparation method described in Example 7, one parameter at a time, were investigated. The changes and their effects are summarised below.

[0426] i) Proportion of the Oil Phase to the Surgihoney-Glycerol Phase

[0427] Oil volumes greater than 10 ml, and less than 10 ml, were tested. The emulsion was found to be more stable when a lower volume of oil was used compared to the volume of the Surgihoney-glycerol phase. When the volume of the oil was less than 6 ml, the emulsion was found to separate by less than 3% in total volume over 72 hours. A volume of 4 ml allowed a separation of just 1.3% of the total volume over 72 hours. This stability is far greater than that of the method described in Example 7, which provided an emulsion with a separation of 9.4% of total volume over the same time period.

[0428] ii) Volume of PGPR

[0429] PGPR volumes up to 4 ml, and less than 1 ml, were tested. The emulsion was more stable when a higher amount of PGPR was used. At a volume of 4 ml, PGPR provided greater stability than with use of lower volumes, and far greater stability than that of the emulsion described in Example 7.

[0430] iii) Shear Rate

[0431] Shear rates from 1000 1/s to 3000 1/s were tested. The emulsion was more stable when a higher shear rate was applied. A shear rate of 3000 1/s produced the most stable emulsion. Separation of only 4.6% of the total volume over 72 hours was observed for emulsion prepared at this shear rate, compared with a separation volume of 9.4% of the total volume over the same time period for emulsion prepared as described in Example 7.

[0432] iv) Temperature

[0433] Temperatures from 20 C. to 40 C. were tested. There was no noticeable trend regard the stability of the emulsions as temperature was increased. However a temperature of 40 C. produced the most stable emulsion. Separation of only 3.1% of the total volume over 72 hours was observed for this emulsion.

[0434] v) Length of Shear

[0435] Shear times of 20 minutes and 30 minutes were tested, in addition to that used in the preparation method described in Example 7. However, there was no significant difference produced by extending the shear time.

[0436] vi) Order of Reagent Addition

[0437] The effect of changing the order in which the reagents are added to the rheometer was tested. The effect of adding all of the components before starting the rheometer was compared with the effect of adding the Surgihoney-glycerol and oil components first, then adding the PGPR after 1-2 minutes. The most stable emulsion was formed when the PGPR was added last. The resulting emulsion provided a separation volume of 2.8% of the total volume over 120 hours.

[0438] vii) Concentration of Surgihoney Dissolved in Glycerol

[0439] The following ratios of Surgihoney (g) to glycerol (ml) were tested: 1 g:1 ml; 0.5 g:1 ml; 2 g:1 ml. The ratio that produced the most stable emulsion was 1 g:1 ml, the same ratio used in the preparation method described in Example 7.

[0440] viii) Sodium Chloride

[0441] When sodium chloride is dissolved in the polar layer of the emulsion, it increases the polarity of this layer. It also forms electrostatic interactions with the lipid layer of the emulsion. The electrostatic interactions and increased polarity could improve stability and reduce coalescence. However, addition of sodium chloride (1 g, 2 g or 4 g) was not found to influence the stability of the emulsion.

[0442] The effects of the changes are summarised in the table below:

TABLE-US-00012 TABLE 8 Stability Shear (% total SH Glycerol Paraffin PGPR rate Temp. Order of vol. after Emulsion (g) (ml) Oil (ml) (ml) (1/s) ( C.) addition 72 hrs) Ex 7 10 10 10 1 2000 37.5 Oil, then 9.4 PGPR, then SH/glycerol Ex 8 (i) 10 10 6 1 2000 37.5 Oil, then 2.8 PGPR, then SH/glycerol Ex 8 (i) 10 10 4 1 2000 37.5 Oil, then 1.3 PGPR, then SH/glycerol Ex 8 (ii) 10 10 10 4 2000 37.5 Oil, then 2.7 PGPR, then SH/glycerol Ex 8 (iii) 10 10 10 1 3000 37.5 Oil, then 4.6 PGPR, then SH/glycerol Ex 8 (iv) 10 10 10 1 2000 40.0 Oil, then 3.1 PGPR, then SH/glycerol Ex 8 (vi) 10 10 10 1 2000 37.5 SH/glycerol 2.8* and oil, then PGPR *(after 120 hrs)

EXAMPLE 9

[0443] Surgihoney Emulsions with High Stability

[0444] The results from the changes described in Example 8 were used to design a further method of preparing Surgihoney emulsion. This method is described below.

Preparation

[0445] 10 g Surgihoney was dissolved in 10 ml of glycerol. 4, 6, 8, or 10 ml of Paraffin oil was then added to the rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 10 ml of Surgihoney-glycerol solution was then added to the rheometer. The rheometer was then started under the following conditions; Shear rate 3000 1/s, Temperature 40 C., gap 4000 m, Run time 10 minutes. After 1 minute 4 ml of PGPR (Polyglycerol polyricinoleate) was then added. Once a total of 10 minutes had elapsed the emulsion was transferred from the rheometer to a container.

TABLE-US-00013 TABLE 9 Surgihoney- Total Total Glycerol Paraf- Volume Volume Formu- (ratio - fin Separation < Separation after lation 1 g:1 ml) PGPR oil 11 Days 20 days number (ml) (ml) (ml) (%) (%) 1 10 4 10 0 0.7 2 10 4 4 0 0.9 3 10 4 4 0 1.0 4 10 4 4 0 1.6 5 10 4 6 0 1.2 6 10 4 8 0 0.9

[0446] All of the formulations were found to be highly stable, with a slight increase in stability observed as the volume of paraffin oil used was increased.

EXAMPLE 10

Surgihoney Cream Formulation

[0447] 1.5 g of Surgihoney was dissolved in 1.5 ml of glycerol. 1 g of sodium alginate was then dissolved in the Surgihoney-glycerol solution. Next 10 ml of Paraffin oil was added to the Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 1 ml of PGPR (Polyglycerol polyricinoleate) was then added. The rheometer was then started under the following conditions; Shear rate 2000 1/s, Temperature set at 37.5 C., gap 4000 m, Run time 10 minutes. After 2 minutes, 1.5 ml of the Surgihoney-alginate and glycerol solution was added to the rheometer. After 3 minutes 8 ml of calcium chloride solution was added dropwise to the rheometer. Once a total of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier to a container.

EXAMPLE 11

Non-Aqueous Surgihoney Cream Formulation

[0448] The method described in Example 10 uses water to dissociate calcium chloride into its ions. This could potentially activate the Surgihoney to produce hydrogen peroxide, and limit the stability of the cream formulation. However, we have appreciated that calcium chloride can be dissociated using non-aqueous solvents, such as ethanol or acetic acid. We have also appreciated that glycerol is able to bind to free water. This property allows water to be used to dissolve the alginate, provided sufficient glycerol is present to prevent premature release of hydrogen peroxide.

[0449] The method described below uses ethanol as a solvent for calcium chloride, and glycerol to bind free water in the alginate solution.

[0450] 1 g of sodium alginate is dissolved in 15 ml water. Next 30 ml glycerol is added to the alginate solution and mixed. Then 30 g Surgihoney is then dissolved in the solution. 10 ml of Paraffin oil is then added to the rheometer (TA Instruments AR-G2) which has a Jacket Peltier and vane geometry attached. 10 ml of Surgihoney solution is then added to the rheometer. The rheometer is then started under the following conditions: Shear rate 3000 1/s, Temperature 40 C., gap 4000 m, Run time 10 minutes. After 1 minute 4 ml of PGPR (Polyglycerol polyricinoleate) is added. After 2 minutes 8 ml of non-aqueous calcium chloride solution (1 M Calcium chloride in ethanol) is added dropwise to the rheometer. Once a total of 10 minutes has elapsed, the emulsion is transferred from the rheometer to a container.

[0451] Summary of emulsion formulations in Examples 7-11:

TABLE-US-00014 TABLE 10 NaAlg (g)/ Shear Emulsion/ SH Glycerol Paraffin PGPR CaCl.sub.2 rate Temp. cream (g) (ml) Oil (ml) (ml) (ml) (1/s) ( C.) Order of addition Ex 7 10 10 10 1 2000 37.5 Oil, then PGPR, then SH/glycerol Ex 9 10 10 4, 6, 8, 4 3000 40.0 Oil and or 10 SH/glycerol, then PGPR Ex 10 1.5 1.5 10 1 (aq) 2000 37.5 Oil and PGPR, then SH/glycerol/NaAlg, then CaCl.sub.2 Ex 11 30 30 10 4 3000 40 Oil and (non-aq) SH/glycerol/NaAlg (aq), then PGPR, then CaCl.sub.2 (non- aq)

EXAMPLE 12

[0452] Optimised Formulation without Addition of Water

[0453] An emulsion was optimised in order to achieve greater stability whilst also attempting to keep viscosity as low as possible. The final formulation and the materials used to generate the emulsion can be seen in their percentage volume in Table 11.

TABLE-US-00015 TABLE 11 The percentage volume of materials used in order to create the optimised formulation without the addition of water. Constituents Volume % (v/v) Surgihoney 20 g Tube 5 ml 20.83 Glycerol 5 ml 20.83 Paraffin Oil 10 ml 41.67 Polyglycerol 4 ml 16.67 polyricinoleate (PGPR)

Method 1:

[0454] Surgihoney first dissolved in glycerol and added to the rheometer cup [0455] Paraffin oil added to rheometer cup. [0456] Rheometer started under following conditions; Shear rate 3000 1/s, 40 C., Gap 4000, 2 minutes. [0457] PGPR added. [0458] Rheometer started under following conditions; Shear rate 3000 1/s, 40 C., Gap 4000, 10 minutes. [0459] After time had elapsed contents of cup was transferred to a 30 ml tube.

[0460] Method 1 was used in order to achieve a stable emulsion using the materials from table 11.

[0461] FIG. 5 shows that the emulsion created using Method 1 can produce a product which is stable over a period greater than 1 month. FIG. 5 a) shows the emulsion on the day of formulation (10 Aug. 2016) and FIG. 5 b) shows the formulation as of 3 Oct. 2016.

[0462] Before addition of water to the emulsion, no reactive oxygen is produced. When water is added to the emulsion, reactive oxygen is produced. This is evidenced by a colour change on a hydrogen peroxide test strip.

[0463] The reverse micelles that are produced by the optimised formulation without the addition of water can be seen in FIG. 6. These spherical droplets contain Surgihoney and help to control the release of reactive oxygen from the emulsion.

[0464] The spray that is produced by the optimised formulation without the addition of water created a jet like ejection (See FIG. 7).

EXAMPLE 12

[0465] Formulation with Addition of Water

[0466] Small amounts of glycerol were added to the formulation. The amount of free water that can be added to the formulation is limited by the amount of glycerol present (1.7 g of glycerol needed to bind to 1 g of water). In order to make sure that reactive oxygen is not produced prematurely, a maximum of 2 mL of water was added to the formulations as the current volume of the emulsions being created only contain 5 mL of glycerol.

TABLE-US-00016 TABLE 12 The percentage volume of materials used in order to create Emulsion 1 which contains 1 ml of water. Constituents of Emulsion 1 Volume % (v/v) Surgihoney RO Tube 4 ml 16.67 Glycerol 5 ml 20.83 Paraffin Oil 10 ml 41.67 Polyglycerol 4 ml 16.67 polyricinoleate (PGPR) Water 1 ml 4.17

TABLE-US-00017 TABLE 13 The percentage volume of materials used in order to create Emulsion 2 which contains 2 ml of water. Constituents of Emulsion 2 Volume % (v/v) Surgihoney RO Tube 3 ml 12.5 Glycerol 5 ml 20.83 Paraffin Oil 10 ml 41.67 Polyglycerol 4 ml 16.67 polyricinoleate (PGPR) Water 2 ml 8.33

[0467] Method 2 is based upon method 1 and continues to use many of the parameters that were optimised previously. The main difference between method 1 and method 2 is that there is an additional step at the beginning. This step is to take into account the addition of free water and aims to stop the premature production of hydrogen peroxide ensuring that the free water is bound to the glycerol prior to the addition of Surgihoney to the system.

Method 2:

[0468] Glycerol and water added to the rheometer [0469] Rheometer started under following conditions; Shear rate 3000 1/s, 40 C., Gap 4000, 2 minutes [0470] Surgihoney and Paraffin oil added to the rheometer [0471] Rheometer started under following conditions; Shear rate 3000 1/s, 40 C., Gap 4000, 2 minutes [0472] PGPR added to the rheometer [0473] Rheometer started under following conditions; Shear rate 3000 1/s, 40 C., Gap 4000, 10 minutes [0474] After time had elapsed contents of cup was transferred to a 30 ml tube.

[0475] FIG. 8 demonstrates the effect that the addition of water has on the stability of the emulsion. In FIG. 8 tubes B and C simply had 1 and 2 mL of water added, respectively to the formulation using method 2. It was found that by adding water without changing the formulation caused instability (phase separation) as can be seen in FIG. 4 b), which was taken after 26 days. By altering the formulation so that the volume of the polar phase is kept the same within the emulsion it was found that the emulsion was more stable. This was achieved by reducing the amount of Surgihoney in the emulsion by the amount of water that is added. This can be seen in tubes D and E in which the amount of Surgihoney within the formulation has been decreased relative to the amount of water added. It was found that with all of these formulations no reactive oxygen was generated prematurely; however all displayed the ability to produce reactive oxygen once excess water was added. These new emulsions containing water were compared to the previously optimised formulation which did not contain water. The emulsions in tube D and E were investigated further and were named emulsions 1 and 2 as this described the amount of water present in each emulsion.

[0476] Two different techniques were used, as demonstrated in in FIGS. 9 and 10; -computed tomography and optical microscopy. These techniques were used in order to investigate the size and distribution of the micelles that were produced. The droplets that can be seen in both figures contain Surgihoney RO and it is the dispersion of these droplets within the continuous phase that controls the release of reactive oxygen in comparison to the raw product.

EXAMPLE 13

Viscosity

[0477] The rheometer (AR-G2 TA Instruments) was set to perform constant shear at rates of 100 and 1000 1/s. The temperature was fixed at 20 C. The samples were assessed about 24 hours after their formulation.

[0478] At lower shear rates it was found that the emulsions which contained water were less viscous than the emulsions which did not contain water, this is evident in FIG. 11 where the viscosities were measured at a shear rate of 100 1/s. However when a high shear rate is applied, the viscosities of the control emulsion and emulsion 1 were found to be similar but the viscosity of emulsion 2 was found to be significantly less. This is demonstrated at a shear rate of 1000 1/s in FIG. 11. Both the control emulsion and emulsion 1 displayed a reduction in viscosity as the shear rate was increased from 100 1/s to 1000 1/s, but the viscosity of emulsion 2 did not differ greatly between these rates.

[0479] The rheometer (AR-G2 TA Instruments) was set to perform a temperature ramp of +2 C. per minute between 10 and 50 C., under a constant shear of 100 1/s.

[0480] The emulsions were created about 24 hours prior to testing. It was found that the control emulsion which contained no water was the most viscous, producing a viscosity of 0.5 Pa.Math.s at 10 C. reducing to a viscosity of 0.07 Pa.Math.s at 50 C. In comparison, Emulsion 2, which contained 2 mL of water, produced the least viscous emulsion with its viscosity at 10 C. being measured at 0.2 Pa.Math.s and dropping to 0.06 Pa.Math.s at 50 C. All three emulsions, as the temperature neared 50 C. produced similar viscosities.

EXAMPLE 14

Release of Hydrogen Peroxide

[0481] In order for an assay to provide accurate results the level of hydrogen peroxide produced must fall within the range of the assay (no greater than 10 M). In order to determine the dilution required for the Surgihoney to produce hydrogen peroxide within this range, values were taken at a number of dilutions. The assay used in this work was a fluorometric hydrogen peroxide assay produced by Sigma Aldrich (MAK165).

[0482] Surgihoney both from the tube and the sachet were allowed to incubate in water for a period of 30 minutes. The assay reagents were then added and incubated for a further 30 minutes before the relative fluorescence units were recorded. This experiment suggested that at dilutions of 1 g/L or less the level of hydrogen peroxide falls within the range of the assay.

[0483] If the concentration of Surgihoney in solution is set at a fixed concentration of 1 g/100 ml the amount of hydrogen peroxide produced in both Emulsions 1 and Emulsions 2 after 30 minutes incubation in water and a further 30 minute incubation in assay reagent, was significantly lower than that of the Surgihoney tube and sachet (See FIG. 13).

[0484] This suggests that there will be a more controlled release over a longer time period in comparison to using the Surgihoney tube and sachet.

EXAMPLE 15

Synthetic Honey Compositions

[0485] Samples with batch number RO contain no glucose oxidase.

[0486] Samples with batch number RO1 contain 50 ppm glucose oxidase.

[0487] Samples with batch number RO2 contain 1000 ppm glucose oxidase.

A. pH 4.03 Buffered Samples

[0488]

TABLE-US-00018 A1. Batch no NB01p43RO Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

[0489] Non sterile base buffered saccharide solution.

TABLE-US-00019 A2. Batch no NB01p43RO Sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

[0490] Sterile base buffered saccharide solution

TABLE-US-00020 A3. Batch no NB01p44RO1 Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

[0491] Non sterile base buffered RO1 saccharide solution.

TABLE-US-00021 A4. Batch no NB01p44RO1 Sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

[0492] Sterile base buffered RO1 saccharide solution

TABLE-US-00022 A5. Batch no NB01p44RO2 Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

[0493] Non sterile base buffered RO2 saccharide solution.

TABLE-US-00023 A6. Batch no NB01p43RO2 Sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0% GOX enzyme N/A

[0494] Description Sterile base buffered RO2 saccharide solution

B. Unbuffered Samples

[0495]

TABLE-US-00024 B1. Batch no NB01p51RO Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0496] Non sterile base buffered saccharide solution.

TABLE-US-00025 B2. Batch no NB01p51RO Sterile Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

[0497] Description Sterile base buffered saccharide solution

TABLE-US-00026 B3. Batch no NB01p51RO1 Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0498] Non sterile base buffered RO1 saccharide solution.

TABLE-US-00027 B4. Batch no NB01p51RO1 Sterile Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0499] Sterile base buffered RO1 saccharide solution

TABLE-US-00028 B5. Batch no NB01p51RO2 Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0500] Non sterile base buffered RO2 saccharide solution.

TABLE-US-00029 B6. Batch no NB01p51RO2 Sterile Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0501] Sterile base buffered RO2 saccharide solution

[0502] C. pH 7.04 buffered samples

TABLE-US-00030 C1. Batch no NB01p57RO Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

Description

[0503] Non sterile base buffered saccharide solution.

TABLE-US-00031 C2. Batch no NB01p57RO Sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

Description

[0504] Sterile base buffered saccharide solution

TABLE-US-00032 C3. Batch no NB01p57RO1 Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

Description

[0505] Non sterile base buffered RO1 saccharide solution.

TABLE-US-00033 C4. Batch no NB01p57RO1 Sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

Description

[0506] Sterile base buffered RO1 saccharide solution

TABLE-US-00034 C5. Batch no NB01p57RO2 Non sterile Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

Description

[0507] Non sterile base buffered RO2 saccharide solution.

TABLE-US-00035 C6. Batch no NB01p57RO2 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

Description

[0508] Sterile base buffered RO2 saccharide solution

EXAMPLE 16

Efficacy of Synthetic Honey Compositions Against Planktonic MRSA

[0509] MIC and MBC were assessed for the RO1 samples (containing 50 ppm glucose oxidase) and compared to Surgihoney (also containing 50 ppm glucose oxidase). See Andrews J. M. Journal of Antimicrobial Chemotherapy (2001) 48, suppl. S1, 5-16.

[0510] The results are shown in FIGS. 14 to 20.

[0511] The results show that, like Surgihoney, synthetic compositions containing glucose, glucose oxidase and fructose are able to inhibit microbial growth.

[0512] Out of all of synthetic compositions, the synthetic composition buffered at pH7.04 had the most effective MIC. Sterilised compositions were more effective than non-sterilised compositions, and the synthetic composition buffered at pH7.04 had the most effective MBC when compared to the other synthetic compositions and even when compared to Surgihoney.

[0513] The results show that, like Surgihoney, synthetic compositions containing glucose, glucose oxidase and fructose are able to inhibit microbial growth.

[0514] FIG. 19 (a to d) and 20 show MIC and MBC results including SurgihoneyRO (RO2) samples and synthetic RO2 samples.

[0515] The synthetic compositions could replace the Surgihoney in the emulsion compositions exemplified in Examples 1 to 14, and also in Examples 16 to 18.

EXAMPLE 16

Modified Methods for Forming Emulsions

[0516] Methods may be modified in order to form emulsions containing smaller micelles. Smaller micelles may lead to an improvement in stability. The size of micelles may be reduced by using a high speed disperser/homogeniser, such as a IKA T 18 digital ULTRA-TURRAX with S 18 N-19 G element.

[0517] A generalised method to create emulsions with small micelles (typically with a range of 10-50 m), is as follows:

1) Pre-mixing to disperse the surfactant (PGPR) in the oil phase (paraffin). The surfactant is added to the oil, the homogeniser (set at 10,000 RPM) is then allowed to run for 2 minutes to ensure adequate dispersion.
2) The Surgihoney (this could include a pre-mix of Surgihoney+Solvent e.g. glycerol) is then slowly added at a rate of 1 ml per 30 seconds to the oil phase.
3) Once Surgihoney is added, the homogeniser is allowed to run until a total elapsed time of 10 minutes (not including pre-mix steps) has been reached.
4) The final emulsion is then transferred from the glass beaker into a sealed tube for storage.

EXAMPLE 17

Modified Method for Forming a Cream

[0518] 0.5% of xanthan gum was added to glycerol at ambient temperature, then heated and moderately mixed to 85 C. 0.1% of magnesium sulfate heptahydrate was then added to the hot mixture and mixed until complete dissolution. The mixing is continued without heating until the mixture reaches 25 C. At this point SurgihoneyRO was added and mixed in a ratio of 2 g SurgihoneyRO: 1.5 ml mixture. Creams of 20 ml were prepared. 10 ml paraffin oil and 2% PGPR (0.4 ml) were added to a beaker and mixed for 1 minute at 10,000 rpm using an IKA ULTRA-TURRAX homogenizer. 10 ml of SurgihoneyRO/solvent/xanthan gum/magnesium sulfate heptahydrate solution was added dropwise for 4 minutes while mixing. The cream was mixed for an extra 5 minutes to reach 10 minutes mixing in total.

EXAMPLE 18

Modified Emulsions and Properties

Method:

[0519] SurgihoneyRO: Paraffin emulsions were prepared in ratios of 30:70, 40:60, 50:50 and 60:40. Paraffin oil and 0.5%, 1% and 2% PGPR were added to a beaker and mixed for 1 minute at 10,000 using an IKA ULTRA-TURRAX homogenizer (Sigma Aldrich Co Ltd, UK). SurgihoneyRO at the corresponding ratio was added dropwise at a rate of 30 seconds per ml while mixing. The emulsion was continued to be mixed until 10 minutes of total mixing time had been achieved.

[0520] Emulsions were created using a T18 Ultra Turrax disperser (IKA, UK). An AR-G2 Rheometer (TA Instruments, UK) was used to determine the viscosity of the emulsions. Droplet size was characterised by use of a Malvern 3000 Mastersizer (Malvern Instruments, UK). The presence of hydrogen peroxide was detected using Quantofix test sticks (Sigma Aldrich, UK) as well as a fluorescence hydrogen peroxide assay (Sigma Aldrich, UK).

[0521] FIG. 21a shows the stability over time of the emulsions with 2% PGPR. FIG. 21b shows the stability over time of the emulsions with 0.5% and 1% PGPR.

[0522] FIG. 22 shows the viscosity at varying shear rates of the emulsions containing 2% PGPR after 7 days.

[0523] FIG. 23 shows G and G measurements of emulsions containing 2% PGPR after 7 days.

[0524] FIGS. 24A and B show how compositions containing 2% PGPR maintained viscoelastic properties after 7 days.

[0525] FIG. 24C showed similar micelle size distributions on day 0 and day 7 in compositions containing 2% PGPR. The average reverse micelle size over this time period was 4.84 m, with a variation in size of between 0.56 m and 22.39 m.

[0526] The formulation also maintained its capacity to generate ROS when stored under ambient conditions (21 C.).

[0527] FIG. 24D shows that a sustained release of hydrogen peroxide occurred over a period of 24 hours.