ANTIMICROBIAL FIBERS AND COMPOSITIONS
20200323963 ยท 2020-10-15
Inventors
- Paulo Bartolo (Southmoor, Abingdon, GB)
- Carl Diver (Southmoor, Abingdon, GB)
- Ian Staples (Southmoor, Abingdon, GB)
- Annette Callaghan (Southmoor, Abingdon, GB)
- Iain Elder (Southmoor, Abingdon, GB)
- Matthew Dryden (Southmoor, Abingdon, GB)
- David Kershaw (Southmoor, Abingdon, GB)
- Rami Salib (Southampton, GB)
Cpc classification
A61L15/26
HUMAN NECESSITIES
A61K38/443
HUMAN NECESSITIES
A61K47/34
HUMAN NECESSITIES
A61K47/46
HUMAN NECESSITIES
D01F6/625
TEXTILES; PAPER
A61L15/40
HUMAN NECESSITIES
International classification
A61K47/34
HUMAN NECESSITIES
A61K47/46
HUMAN NECESSITIES
A61K9/70
HUMAN NECESSITIES
A61L15/26
HUMAN NECESSITIES
A61L15/40
HUMAN NECESSITIES
Abstract
Fibers for generating antimicrobial activity are described. The fibers comprise an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substance that includes a substrate for the enzyme. In the presence of sufficient free water, the enzyme converts the substrate to release hydrogen peroxide, which is effective against a wide range of microbes. Wound dressings and compositions for forming such fibers are also described, as is the use of the fibers, wound dressings and compositions.
Claims
1. An electrospinnable composition comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, a substance that includes a substrate for the enzyme, an electrospinnable polymer and a non-aqueous solvent.
2. A composition according to claim 1, wherein the electrospinnable polymer is polycaprolactone.
3. A composition according to claim 1 or claim 2, wherein the non-aqueous solvent is, or comprises, an acidic solvent, preferably acetic acid.
4. A composition according to any preceding claim, comprising 5-30%, by weight, of the electrospinnable polymer.
5. A composition according to any preceding claim, comprising 10-30%, by weight, of the electrospinnable polymer.
6. A composition according to any preceding claim, comprising about 20%, by weight, of the electrospinnable polymer.
7. A composition according to any preceding claim, comprising 0.5-50%, by weight, of the substance.
8. A composition according to any preceding claim, comprising 5-40%, by weight, of the substance.
9. A composition according to any preceding claim, comprising 5-30%, by weight of the substance.
10. A composition according to any of claims 1 to 6, comprising 10 to 60%, by weight, of the substance, preferably 20 to 50%, by weight, of the substance.
11. A composition according to any preceding claim, in which the enzyme is a purified enzyme.
12. A composition according to any preceding claim, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance.
13. A composition according to any preceding claim, in which the substance lacks catalase activity.
14. A composition according to any preceding claim, in which the substance is an unrefined natural substance.
15. A composition according to any preceding claim, in which the substance is a sugar substance.
16. A composition according to any preceding claim, wherein the substance is honey.
17. A composition according to claim 16, wherein the honey is an unpasteurised honey, preferably a creamed unpasteurised honey.
18. A composition according to claim 16, wherein the honey is a pasteurised honey.
19. A composition according to any preceding claim, wherein the enzyme is an oxidoreductase enzyme, preferably a glucose oxidase.
20. A composition according to any of claims 1 to 13, wherein the substance comprises a purified substrate for the enzyme.
21. A composition according to any preceding claim which does not Include sufficient free water to allow the enzyme to convert the substrate.
22. A fiber obtained or obtainable by electrospinning a composition according to any preceding claim.
23. A fiber according to claim 22, obtained or obtainable by electrospinning at a flow rate of 0.2 to 5 ml/min.
24. A fiber according to claim 22 or claim 23, obtained or obtainable by electrospinning at a flow rate of 0.5 to 1 ml/min.
25. A fiber according to any of claims 22 to 24, obtained or obtainable by electrospinning at a flow rate of about 0.8 ml/min.
26. A fiber according to any of claims 22 to 25, obtained or obtainable by electrospinning at an applied voltage of 5-50 kV.
27. A fiber according to any of claims 22 to 26, obtained or obtainable by electrospinning at an applied voltage of 10-20 kV.
28. A fiber according to any of claims 22 to 27, obtained or obtainable by electrospinning at an applied voltage of about 13 kV.
29. A fiber according to any of claims 22 to 28, obtained or obtainable by electrospinning at a collecting distance of 5-30 cm.
30. A fiber according to any of claims 22 to 29 obtained or obtainable by electrospinning at a distance of 10-20 cm.
31. A fiber according to any of claims 22 to 30, obtained or obtainable by electrospinning at a collecting distance of about 15 cm.
32. A fiber according to any of claims 22 to 31, obtained or obtainable by electrospinning at a processing time of 10 minutes to 100 minutes.
33. A fiber according to any of claims 22 to 32, obtained or obtainable by electrospinning at a processing time of 30 minutes to 60 minutes.
34. A fiber according to any of claims 22 to 33, obtained or obtainable by electrospinning at a processing time of about 40 minutes.
35. A fiber according to any of claims 22 to 34, which does not include sufficient free water to allow the enzyme to convert the substrate.
36. A wound-dressing comprising one or more fibers as defined in any of claims 22 to 35.
37. A fibrous mat comprising one or more fibers as defined in any of claims 22 to 36.
38. A method comprising electrospinning a composition as defined in any of claims 1 to 21.
39. A method according to claim 38, comprising maiming at a flow rate of 0.2 to 5 ml/min.
40. A method according to claim 38 or claim 39, comprising electrospinning at a flow rate of 0.5 to 1 ml/min.
41. A method according to any of claims 38 to 40, comprising electrospinning at a flow rate of about 0.8 ml/min.
42. A method according to any of claims 38 to 41, comprising electrospinning at an applied voltage of 5-50 kV.
43. A method according to any of claims 38 to 42, comprising electrospinning at an applied voltage of 10-20 kV.
44. A method according to any of claims 38 to 43, comprising electrospinning at an applied voltage of about 13 kV.
45. A method according to any of claims 38 to 44, comprising electrospinning at a collecting distance of 5-30 cm.
46. A method according to any of claims 38 to 45, comprising electrospinning at a distance of 10-20 cm.
47. A method according to any of claims 38 to 46, comprising electrospinning at a collecting distance of about 15 cm.
48. A method according to any of claims 38 to 47, comprising electrospinning at a processing time of 10 minutes to 100 minutes.
49. A method according to any of claims 38 to 48, comprising electrospinning at a processing time of 30 minutes to 60 minutes.
50. A method according to any of claims 38 to 49, comprising electrospinning at a processing time of about 40 minutes.
51. A fiber comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, a substance that includes a substrate for the enzyme, an electrospinnable polymer and a non-aqueous solvent.
52. A fiber according to claim 51, which is a nanofiber.
53. A fiber according to claim 52 or claim 53, wherein the electrospinnable polymer is polycaprolactone.
54. A fiber according to any of claims 51 to 53, wherein the non-aqueous solvent is an add, preferably acetic acid.
55. A fiber according to any of claims 51 to 54 wherein the substance is an unrefined natural substance, such as honey.
56. A fiber according to any of claims 51 to 55, which does not include sufficient free water to allow the enzyme to convert the substrate.
57. A fiber according to any of claims 51 to 56, comprising up to 80%, by weight, of the substance.
58. A fiber according to any of claims 51 to 57, comprising up to 60%, by weight, of the substance.
59. A fiber according to any of claims 51 to 58, comprising up to 40%, by weight, of the substance.
60. A fiber according to any of claims 51 to 59, comprising up to 20%, by weight, of the substance.
61. A fiber according to any of claims 51 to 60, comprising up to 80%, by weight, of the polymer.
62. A fiber according to any of claims 51 to 61, comprising up to 60%, by weight, of the substance.
63. A fiber according to any of claims 51 to 62, comprising up to 40%, by weight, of the substance.
64. A fiber according to any of claims 51 to 63, comprising up to 20%, by weight, of the substance.
65. A fiber according to any of claims 51 to 64, wherein the enzyme is a purified enzyme.
66. A fiber according to any of claims 51 to 65, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance.
67. A fiber according to any of claims 22 to 35 or 51 to 88, with a diameter of 0.2 to 20 m.
68. A wound dressing or fibrous mat, comprising one or more fibers as defined in any of claims 51 to 67.
69. A wound dressing or fibrous mat according to claim 68, wherein the mean fiber diameter is 1 m to 10 m.
Description
[0311] Preferred embodiments of the invention, and preferred compositions that can be used in making fibers, dressings and compositions of the invention, are now described, by way of example only, with reference to the accompanying drawings in which:
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EXAMPLE 1
[0353] This example describes a preferred process for production of a storage-stable composition for use in the invention, and dilution of the storage-stable composition to release hydrogen peroxide.
Process for Manufacture of Activated Honey
[0354] Honey is heated for 2 minutes to 80 C. using a heat exchanger (a lower temperature could be used if desired, suitably at least 60 C., provided this is sufficient to inactivate catalase). The purpose of this heating process is to pasteurise the honey, reduce its viscosity so that it can be filtered to remove any wax particles and bee wings that may be in the honey post harvest, and inactivate any catalase in the honey that would affect the efficient production of hydrogen peroxide.
[0355] The pasteurised honey is then filtered, and left to cool naturally to normal hive temperatures (35-40 C.). Glucose oxidase is then added at a low level (equivalent to the level found normally in honey) to replace the glucose oxidase naturally found in the honey but which has been inactivated by the pasteurisation process. The filtered pasteurised honey is fairly liquid at 35-40 C. so it can easily be mixed with the glucose oxidase. There is, however, no other reason why the mix could not be done at room temperature. The resultant activated honey is then stored at ambient temperature.
[0356] Apart from the replacement of the inactivated glucose oxidase there is no modification to the composition of the natural honey. There is no detectable hydrogen peroxide in the activated honey.
Dilution of Activated Honey
[0357] Following dilution of the activated honey, hydrogen peroxide is released after a lapse of time, as free water becomes available and the glucose oxidase starts to convert the glucose present in the honey. The hydrogen peroxide level is less than 2 mmol/litre but is released for an extended period.
EXAMPLE 2
[0358] This example describes the results of tests demonstrating the antimicrobial effect of activated Ulmo honey. The honey is described as activated if it contains added glucose oxidase.
Well Diffusion Assay Staphylococcus aureus (NCIMB 9518) was grown on nutrient agar or in nutrient broth.
[0359] Antibiotic diffusion agar plates were inoculated with culture by swabbing overnight culture onto the surface of agar plates. Plates were allowed to stand at room temperature for 15 minutes.
[0360] Wells 7 mm diameter were bored into the surface of the agar. Two hundred microlitres of sample (phenol standard, or honey) was placed into each well.
[0361] Plates were incubated for 16 hrs and zones of inhibition were measured using a dial calipers (+/0.1 mm). The diameter of zones, including the diameter of the well, were recorded.
[0362] Phenol standards were prepared by diluting phenol in purified water at the required concentration. For example, a 10% phenol standard was prepared by diluting 1 g of phenol in 9 g of purified water. The standards were stored at 30 C. and shaken before use.
Honey
[0363] Honey: Pasteurised Ulmo honey.
[0364] Non-activated honey (i.e. honey to which no glucose oxidase had been added) was used as a control.
Enzyme Preparation
[0365] Glucose oxidase: medical device grade material, non food grade, from GMO Aspergillus niger, supplied by Biozyme UK, activity 240 iu/mg. Initially 0.5% w/w enzyme was used, but this could be reduced to 0.005% w/w enzyme to achieve a 20% phenol standard equivalent.
Detection of Hydrogen Peroxide
[0366] Peroxide test from Merckoquant: Cat. No. 1.10011.0002 Measuring range/colour-scale graduation mg/l H2O2 0.5-2-5-10-25.
[0367] Procedure determination in aqueous solutions: dissolve honey in water 50/50 w/w. Immerse the reaction zone of the test strip in the measurement sample (15-30 C.) for 1 second. Allow excess liquid to run off via the long edge of the strip onto an absorbent paper towel and after 15 seconds (Cat. No. 110011) or after 5 seconds (Cat. No. 110081) determine with which colour field on the label the colour of the reaction zone coincides most exactly. Read off the corresponding result in mgA H.sub.2O.sub.2 or, if necessary, estimate an intermediate value.
[0368] To determine that no endogenous hydrogen peroxide is available in the honey, dissolve in methanol 50/50 w/w. Determination in organic solvents (readily volatile ethers): Immerse the reaction zone of the test strip in the measurement sample (15-30 C.) for 1 second. After the solvent has evaporated (gently fan the strip back and forth for 3-30 seconds) immerse the reaction zone in distilled water for 1 second and allow excess liquid to run off via the long edge of the strip onto an absorbent paper towel or gently blow on the reaction zone four times, for 3-5 seconds each time. After 15 seconds (Cat. No. 110011) or after 5 seconds (Cat. No. 110081) determine with which colour field on the label the colour of the reaction zone coincides most exactly. Read off the corresponding result in mg/l H.sub.2O.sub.2 or, if necessary, estimate an intermediate value.
Results
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[0370] The antimicrobial effect of activated honey containing at least 0.001% w/w glucose oxidase (240 iu/mg) on Staphylococcus aureus was equivalent to that of Manuka 25+ honey.
[0371] The effect of the activated honey is bactericidal.
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[0373] The results show that the effect of the activated Ulmo honey was equivalent to a 30% phenol standard, and was over twice as effective as MGO Manuka honey, and nearly twice as effective as Manuka UMF 25+ honey.
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EXAMPLE 3
[0375] This example describes the results of tests demonstrating the antimicrobial effect of activated Tineo honey. The honey is described as activated if it contains added glucose oxidase.
[0376] Well diffusion assays as described in Example 2 were carried out using samples of TINED honey and different phenol standards.
1.10% Phenol Standard
2.20% Phenol Standard
3.30% Phenol Standard
[0377] 4. TINED Honey (pasteurized TINED honey containing no added glucose oxidase)
5. TINED Dead (pasteurized TINED honey, with further heat deactivation)
6. Manuka UMF 25+
[0378] 7. TINED 20+ (pasteurized TINED honey with added glucose oxidase, 0.005% Biozyme enzyme)
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1.10% Phenol Standard
2.20% Phenol Standard
3.30% Phenol Standard
[0381] 4. TINED Honey Active 25+(0.005% enzyme w/w)
5. TINED Honey Active 25+(0.005% enzyme w/w)
6. TINED Honey Active 40+(0.05% enzyme w/w)
7. Manuka Honey UMF 25+
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EXAMPLE 4
[0384] This example describes the results of tests demonstrating the antimicrobial effect of activated Ulmo and Tineo honey.
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[0388] In this test, 0.005% w/w enzyme was approximately equivalent to the 10% phenol standard, 0.05% w/w enzyme was approximately equivalent to the 15% phenol standard, and 0.5% w/w enzyme was approximately equivalent to the 25% phenol standard. The Manuka 18+ honey was equivalent to the 10% phenol standard, and the Manuka 25+ honey showed activity that was intermediate between the 10% and 15% phenol standard.
EXAMPLE 5
[0389] In this example, the effect of different glucose oxidase enzyme preparations was tested with pasteurized Tineo honey. The enzyme preparations used were the Biozyme preparation as described in Example 2, and a food grade, non-GMO source, glucose oxidase from ANHUI MINMETALS DEVELOPMENT I/E CO., LTD (referred to as the Anhui enzyme preparation below).
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EXAMPLE 6
[0392] This example describes the results of tests of the stability of activated honeys stored for 60 or 90 days.
[0393] Glucose oxidase enzyme from Biozyme, as described in Example 2, was added to pasteurized Ulmo or Tineo honey at 0.005% w/w, and the resulting mixtures were stored at 37 C. for two months (Ulmo honey samples) or at room temperature for 90 days (Tineo honey samples). The amount of enzyme used corresponds to the amount that would be used for a commercial food product.
[0394] After storage, the samples were tested in a well diffusion assay against Staphylococcus aureus carried out as described in Example 2. The stored samples were compared with 10%, 20%, and 30% phenol standards.
EXAMPLE 7
[0395] This example describes the results of tests of the antimicrobial activity of a powdered activated honey in which glucose oxidase enzyme in powder form is added to a powdered honey.
[0396] Powdered honey was obtained from Honi Bake from ADM Specialty Ingredients. 0.1% w/w Biozyme glucose oxidase (as described in Example 2) was added to the powdered honey. The antimicrobial activity of the mixture was tested in a well diffusion assay against Staphylococcus aureus as described in Example 2. The mixture was added to two different wells of the agar plate. A control was used with honey powder only. The results are shown in
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EXAMPLE 8
[0398] This example describes sterilisation of a composition that comprises unpasteurised honey and added purified glucose oxidase.
[0399] Ten sealed sachets each containing 50 g of the composition were gamma irradiated at a target dose of 11.6-14.2 kGy (the dose was 13.1-13.6 kGy as determined by dosimeters), and subsequently individually tested for sterility.
[0400] For the sterility testing, all work was carried out in a cleanroom under a laminar flow. 10 g of the same was added to 100 ml of sterile Tryptone Soya Broth (TSB: pancreatic digest of casein, 17 g/L, papaic digest of soya bean meal, 3 g/L, sodium chloride, 5 g/L, dibasic potassium phosphate, 2.5 g/L, glucose 2.5 g/L, pH 7.30.2) and shaken to mix, then transferred to a sterile container. A further 100 ml of TSB was added to remove any sample residue and added to the same container. TSB was added to the sample and incubated at 30 C.2 C. for a minimum of 14 days and inspected for signs of microbial growth. Positive controls were performed on all media before testing commenced.
[0401] One positive result was noted after the full incubation period. Substantiation of 35 kGy as a sterilization dose was accepted.
[0402] Testing of sachets before and after sterilisation by gamma irradiation using a well diffusion assay similar to the assay described in Example 2 confirmed that the effect of irradiation on the antimicrobial activity of the composition was negligible. There was no observable reduction in activity level following sterilisation.
EXAMPLE 9
[0403] This example describes the results of tests demonstrating the antimicrobial effect of a composition that comprises unpasteurised honey and added purified glucose oxidase (referred to as Surgihoney) that has been sterilised using gamma irradiation, compared with Manuka UMF 25+ honey, and a heat-deactivated honey (Non Active Honey).
[0404] Well diffusion assays as described in Example 2 were carried out using samples of Manuka honey UMF 25+, Non Active Honey, and Surgihoney.
[0405] The results clearly show that Surgihoney retains significant antimicrobial activity against Staphylococcus aureus after sterilisation, and that the Surgihoney was more effective against Staphylococcus aureus than Manuka honey UMF 25+.
EXAMPLE 10
[0406] Stability testing of a composition that comprises unpasteurised honey and added purified glucose oxidase that has been sterilised using gamma irradiation (at a minimum dose of 35 kGy).
[0407] Accelerated aging techniques are based on the assumptions that the chemical reactions involved in the deterioration of materials follow the Arrhenius reaction rate function. This function states that a 10 C. increase or decrease in the temperature of a homogenous process, results in approximately a two times or % time change in the rate of a chemical reaction. For example, at 55 C., 5.3 weeks is equivalent to 1 year on-the-shelf, and at 55 C., two years would be equivalent to 10.6 weeks and five years would be 26.5 weeks.
[0408] Products from two different production batches were used for this study. Products were sachets that contained 10 g of the composition. The sachets had been sterilized at a minimum dose of 35 kGy gamma irradiation. Samples were stored, under accelerated aging conditions, at 55 C. (2 C.).
[0409] The relationship between real time and accelerated ageing is as follows:
TABLE-US-00011 TABLE 2 Real Time equivalent Accelerated Aging @ 55 C. (Months) Days Weeks 3 9 1.3 6 19 2.6 12 37 5.3 24 74 10.6 36 111 15.9 48 148 21.2 60 185 26.5
Testing, Test Intervals And Samples Required
[0410] Samples from each batch we tested at the same time intervals. The following table summarises the tests performed, and the total number of samples required at each time point for each batch.
TABLE-US-00012 TABLE 3 Time Intervals (Real = R; Accelerated = A) all in weeks 26 weeks Test 26R 2.6A Filled sachet weight 10 10 Pressure test 10* 10* pH of honey 5** 5** Moisture level 5** 5** Colour 1 1 Sterility Samples per batch 11 11 Total Samples 33 33 *Use same samples as for filled sachet weight test **Use five samples from pressure test
Test Methods
[0411] Filled sachet weight: An empty sachet has an average tare weight of 1.7 g. 10 sachets were weighed individually on a calibrated laboratory balance, the tare weight was subtracted, and the results were recorded.
[0412] Pressure Test: Each sachet was tested on a Pressure Test Rig in accordance with standard operating procedures. The number of passes and failures was recorded. pH of honey: The contents of five sachets were pooled into a glass beaker. A Hanna pH meter was calibrated using three standard solutions, then the electrode was rinsed in DI water. The pH electrode and temperature probe were immersed into the honey sample and the pH value and temperature were read from the digital display, and recorded.
[0413] Moisture level: Five sachets of honey were taken from each time point. A sample of approximately 1 ml from each sachet was taken and placed on the sample plate of a refractometer (one sample at a time). The sample cover was dosed and the user looked through the lens whilst pointing the instrument at a source of light such as a window. The value of the scale was read, as indicated by the line of shadow, and the result was recorded.
[0414] Colour. One sachet of honey was opened and some of the honey composition was placed onto a white tile. The colour of the honey composition was compared with the Honey Colour Chart Pfund Scale, and the score was recorded (30-800).
Results
Filled Sachet WeightsAccelerated Aging
Batch A:
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TABLE-US-00013 TABLE 4 Test interval Weight of honey in grams - total weight minus tare weight (weeks) 1 2 3 4 5 6 7 8 9 10 2.6 10.3 10.3 10.1 10.2 10.3 10.3 10.2 10.3 10.3 10.2
Batch B:
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TABLE-US-00014 TABLE 5 Test Interval Weight of honey in grams - total weight minus tare weight (weeks) 1 2 3 4 5 6 7 8 9 10 2.6 10.3 12.1 10.3 10.6 10.6 9.8 10.1 10.1 10.1 9.8
Pressure Test ResultsAccelerated Aging
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TABLE-US-00015 TABLE 6 Test interval (weeks) Batch A Batch B 2.6 10/10 10/10
pH Test ResultsAccelerated Aging
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TABLE-US-00016 TABLE 7 Test interval Batch A Batch B (weeks) pH Temp ( C.) pH Temp ( C.) 2.6 3.8 23.1 3.71 23.0
Moisture Content Test ResultsAccelerated Aging
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TABLE-US-00017 TABLE 8 Test interval Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 (weeks) (%) (%) (%) (%) (%) 2.6 15.8 15.8 15.8 15.8 15.6
Batch A:
Batch B:
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TABLE-US-00018 TABLE 9 Test interval Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 (weeks) (%) (%) (%) (%) (%) 2.6 15.6 15.8 15.6 16.2 16.2
Colour Test ResultsAccelerated Aging
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TABLE-US-00019 TABLE 10 Test interval Pfund scale score (weeks) Batch A Batch B 2.6 90-120 90-120
[0422] Examples 11-19 below describe the results of treatment of wounds using a composition comprising unpasteurised honey with added glucose oxidese (the composition is referred to in the examples as Surgihoney). The Surgihoney was provided in sealed sachets, each containing 10 g of the composition. The sachets had been sterilised using gamma irradiation. Sachets were used from day 0 of treatment. Each dressing change involved a fresh application of Surgihoney. The dressing was changed at each of the days recorded in the example, or sometimes more frequently. The Surgihoney was applied to a dressing, or directly to the wound, and then covered by a dressing. In both cases the Surgihoney was in direct contact with the wound and was covered by a dressing.
EXAMPLE 11
[0423] This example describes the results of treatment of an infected toe using Surgihoney. The results are shown in
[0424] The patient was a 78 year old diabetic male. The wound on the left foot developed over the month before treatment began and was causing mild pain. [0425] a) Day 0Wound Profile: wound: 321 cm; 99% Healthy granulation but 1% green colonisation; surrounding skin excoriated; small amount of yellow pus exudate giving off an odour; [0426] b) Day 5Wound Profile: Wound improved; Second dressing change with 1 honey sachet applied each time; Metrondiazole & amoxicillin; Also Tea tree oil applied; [0427] c) Day 10Wound Profile: Wound Improved; Green colonisation gone; Wound cleaned, dry skin removed and further sachet of honey applied
EXAMPLE 12
[0428] This example describes the results of treatment of a toe ulcer using Surgihoney. The results are shown in
[0429] The patient was a 61 year old diabetic female with an infected toe ulcer on the right foot developing over a month-long period prior to treatment, and causing mild pain. [0430] a) Day 0Wound Profile: Wound: 220.2 cm; 1% yellow brown slough, rest granulated tissue; Low amount of yellow exudate; [0431] b) Day 7Wound Profile: Wound improved; Daily application on 0.5 sachets of Surgihoney with dressing change; Flucox; [0432] c) Day 10Wound Profile: Wound improved further, Flucox stopped.
EXAMPLE 13
[0433] This example describes the results of treatment of a foot ulcer using Surgihoney. The results are shown in
[0434] The patient was a 50 year old female diabetic with a foot ulcer that had developed the month before treatment. Fragile skin but no pain was reported. [0435] a) Day 0Wound Profile: Wound: 10.50.5 cm; 1% yellow brown slough, rest granulated tissue; Very low amount of low, yellow exudates; [0436] b) Day 7Wound Profile: Wound improved; Dry; slough replaced by healthy granulation; Size and depth reduced; Wound cleaned with saline and then honey dressing (0.5 sachet) applied; Ni antibiotics; [0437] c) Day 9Wound Profile: Wound much Improved; Needy dosed with no exudates present; Dressings continue to be applied with 0.5 sachets on each.
EXAMPLE 14
[0438] This example describes the results of treatment of a diabetic foot ulcer using Surgihoney. The results are shown in
[0439] The patient was a male with a diabetic foot ulcer caused by poor quality shoe irritation. The patient had the ulcer less then 1 month prior to treatment. [0440] a) Day 0Wound Profile: Red surrounding skim with mild pain; 1% slough, 99% granulated; Infection and diabetes; Low volume exudate, serous and yellow; Wound size: 2 cm2 cm0.2 cm; [0441] b) Day 7Wound Profile: Assessment done in hospital but the dressing had been changed daily from day 1 until now. The dressings were changed according to the protocol by daughter in law, who is a trained nurse. Wound improved; 100% healthy granulation; Healthy surrounding skin, mild pain; Antibiotics used, Flucoxacillin, 500 mg, every 6 hours for 7 days; Wound size: 1.5 cm1.3 cm0.1.
EXAMPLE 15
[0442] This example describes the results of treatment of a traumatic leg wound using Surgihoney. The results are shown in
[0443] The patient was a 95 year old female with a traumatic wound to the lower leg that caused moderate pain. [0444] a) Day 0Wound Profile: Wound: 15121 cm; Mostly granulitic tissue but with 1% of wound bed necrotic and black; Medium amount of haemoserous exudate; [0445] b) Day 3Wound Profile: Wound static; No further necrosis but wound much the same; 1 sachet of honey applied; Swab: Entrecoccus sp.; nil antibiotics; [0446] c) Day 8Wound Profile: Wound improved; Lower volume of exudate and reduced necrotic tissue.
EXAMPLE 16
[0447] This example describes the results of treatment of an infected leg wound using Surgihoney. The results are shown in
[0448] The patient was a 91 year old female with an infected wound to the lower leg. Surrounding skin was fragile and mild pain existed. [0449] a) Day 0Wound Profile: Wound Size: 4.52.5 cm; Mostly red granulitic tissue with 1% yellow/brown slough; Medium volume yellow serous exudate. [0450] b) Day 15Wound Profile: Wound improved; Size: 42 cm; Less slough and serous exudate present; [0451] c) Day 19Wound Profile: Wound improved; Surgihoney applied with each dressing.
EXAMPLE 17
[0452] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0453] The patient was a female with a Leg Ulcer that had developed over the year before treatment began, and caused mild pain. [0454] a) Day 0Wound Profile: Healthy surrounding skin; 1% slough, 99% granulated; Medium volume exudate, serous and yellow; Wound size: 7 cm3 cm; [0455] b) Day 7Wound Profile: Wound improved and patient in community care; 1 sachet of Surgihoney applied; Size reduced and less exudate present Pain more bearable.
EXAMPLE 18
[0456] This example describes the results of treatment of a pressure ulcer using Surgihoney. The results are shown in
[0457] The patient was an 88 year old female diabetic with poor nutritional status. The patient had a pressure ulcer (grade 3) that had developed over the 6 month period prior to treatment, causing a moderate level of pain with fragile surrounding skin. [0458] a) Day 0Wound Profile: Wound size: 0.70.70.2 cm; 1% yellow/brown slough, 1%; cellulite tissue with rest granulitic; Low volume of yellow pus present; [0459] b) Day 3Wound Profile: Wound healed (closed up); 1 Sachet of Surgihoney applied.
EXAMPLE 19
[0460] This example describes the results of treatment of infection surrounding the entry point of a catheter using Surgihoney. The results are shown in
[0461] The patient was a 41 year old female cancer patient. The patient had infection of breast area surrounding entry point of catheter. The wound was less than 1 week old prior to treatment mild pain caused. [0462] a) Day 0Wound Profile: Wound: 2 cm2 cm; 1% cellultic, rest granulated tissue; Low volume of exudate, red and serous; [0463] b) Day 6Wound Profile: Wound much improved; No exudates; One sachet of surgihoney applied; Swab taken but no growth; nil antibiotics; [0464] c) Day 14Wound Profile: Wound improved to the extent that it is no longer an issue.
EXAMPLE 20
Surgihoney
[0465] Surgihoney is unpasteurised honey with added purified glucose oxidase. Three different preparations of Surgihoney were made with different antimicrobial potencies:
S1 Surgihoney (Also Referred to as SH1):
[0466] unpasteurised honey with 0.1% (w/w) added glucose oxidase. The enzyme used was food grade glucose oxidase, from Aspergillus niger, from BIO-CAT, INC, activity 15,000 Units/g. Sealed sachets containing 50 g of the S1 Surgihoney were gamma irradiated at a target dose of 11/6-14.2 kGy.
S2 Surgihoney (Also Referred to as SH2):
[0467] unpasteurised honey with 0.1% (w/w) added glucose oxidase. The enzyme used was glucose oxidase (GO3B2), from Aspergillus niger, from BBI Enzymes Limited, activity 274 Units/mg. Unit Definition: the amount of enzyme causing the oxidation of 1 micromole of glucose per minute at 25 degrees centigrade at pH 7.0. Contaminants: alpha amylase no greater than 0.05%, Saccharase no greater than 0.05%, maltase no greater than 0.05% and GO/Cat no less than 2000.
S3 Surgihoney (Also Referred to as SH3):
[0468] unpasteurised honey with 0.25% (w/w) added glucose oxidase. The enzyme used was glucose oxidase (GO3B2) from BBI Enzymes Limited, activity 274 Units/mg.
[0469] Thus, S1 Surgihoney contains 15 units of glucose oxidase per gram of the composition, S2 Surgihoney contains 274 units of glucose oxidase per gram of the composition, and S3 Surgihoney contains 685 units of glucose oxidase per gram of the composition.
EXAMPLE 21
In Vitro Antimicrobial Activity of Surgihoney
[0470] This example describes susceptibility testing of a range of wound and ulcer bacterial isolates to Surgihoney by disc diffusion method, minimum inhibitory concentration (MIC) and minimum cidal concentration (MBC) determination, and time bactericidal measurements.
Summary
Results:
[0471] Surgihoney demonstrates highly potent Inhibitory and tidal 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. Topical concentration of Surgihoney in wounds is estimated at approximated 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. 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.
Conclusions:
[0472] 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 for skin lesions, wounds, ulcers and cavities. It is highly active against multidrug resistant bacteria. It Is more active than other honeys tested and comparable to chemical antiseptics in antimicrobial activity.
[0473] Superficial wounds and skin ulcers are becoming increasingly common with the rising age of the population in many countries and the global epidemic of obesity and type 2 diabetes. In the UK, community nurses spend much of their time dressing leg ulcers and supervision by leg ulcer nurses is essential if standards are to be maintained in community leg ulcer services. Most chronic breaks in the skin become colonised with bacteria. It is difficult to know when and if these are pathogenic but it is likely that even if overt infection is not present, bacterial colonisation plays a role in slowing tissue healing, establishing biofilm and resulting in wound slough and an offensive odour.
[0474] Tissue viability Is challenging particularly when complicated by comorbidities. Chronic wounds always become colonised with bacteria which may destabilise the healing process. There is a temptation to send a microbiological sample and to offer systemic antibiotics when the sample is reported as growing bacteria. All this serves is to select ever more resistant microbes which is why chronic lower extremity ulcers are so often colonised with multidrug resistant organisms such as methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.
[0475] Surgihoney has been developed as a prophylactic dressing for wounds. 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
[0476] 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.
[0477] Clinical isolates were collected from soft tissue microbiology samples. Eighteen isolates of Staphylococcus aureus, 12 meticillin-sensitive (MSSA) and 6 meticillin-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
[0478] 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.
[0479] A pilot study was carried out initially to compare Surgihoney potencies S1, S2, S3 with a variety of honeys from around the world, European, South American, New Zealand, Yemeni, 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.
[0480] Following the pilot studies the Surgihoney potencies S1, 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 app., and anaerobically for Propionibacterium sp. And Bacteroides sp.)
[0481] Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations 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.
[0482] 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.
[0483] All web 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.
[0484] The MIC well and those around the MIC well we 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
[0485] 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.
[0486] The test and control inocula we 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.
[0487] 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.
[0488] 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).
[0489] 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 11. 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 11.
MIC's & MBC's
[0490] Surgihoney demonstrated significant antimicrobial activity against all the isolates tested. MIC's and MBCs we very consistent amongst isolates of the same species whether the isolates were multidrug resistant or highly sensitive. Table 12 lists the MIC and MBC values for isolate species tested by dilution ratio and Table 13 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.
[0491] Topical concentration of Surgihoney in wounds 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.
[0492] 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 (
Discussion
[0493] 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.
[0494] 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.
[0495] 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 in any colonised or superficially infected wounds or soft tissue cavities. As many chronic wounds are colonised with resistant bacteria, and bacterial persistence in biofilm production delays wounds healing, Surgihoney use may help reduce in appropriate use of antibiotics as well as promote wound healing. In clinical use, the topical Surgihoney concentrations at the site of the wound will be considerably higher than those for systemic antibiotics in serum or deep tissue. This is reflected in the values of the MIC and MBC's for Surgihoney, which are correspondingly higher than those generally expressed for systemic antibiotics.
[0496] 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.
[0497] Surgihoney is formulated as a sterile product to be applied as a topical wound dressing to skin lesions and cavities with the aim of providing a moist wound healing environment whilst also reducing microbial colonisation, helping to remove slough and to promote granulation and epithelialisation.
[0498] Other antimicrobial preparations are available as topical preparations intended to treat or prevent wound infections. Silver impregnated dressings appear to possess good antimicrobial activity however they also display cytotoxicity compared to honey preparations. Iodine analogues also possess good antimicrobial activity but they also have been reported to be toxic in certain situations. There is also increasing concern about the use of chlorhexidine preparations in wound dressings due to the development of antimicrobial resistance and toxicity.
[0499] The clinical utility of Surgihoney is likely to be in topical application, on skin, in wounds and cavities. Wounds may become colonised with bacteria which can form biofilms and delay healing. With increasing concern about antimicrobial resistance and the lack of novel antimicrobial agents, a topical agent with broad antimicrobial activity, could play a role in reducing the use of systemic antibiotics in soft tissue lesions. These in vitro studies have demonstrated the potential of Surgihoney as a wound dressing with high antimicrobial activity whose potency can be controlled and which also delivers other important functions in wound healing: moist barrier, desloughing, local nutrient supply, local immune modulation and is not cytotoxic.
Conclusion
[0500] These in vitro results support the clinical use of Surgihoney as a wound dressing and this may be the first product that can deliver all the required roles in the healing process of wounds as well as being a potent and non-toxic antimicrobial.
TABLE-US-00020 TABLE 11 Inhibitory zones sizes with different potencies of Surgihoney (S1, S2, S3) No. S1 Mean S2 Mean S3 Mean of zone zone 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 1 32.1 43.7 55.2 lwoffii 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-00021 TABLE 12 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-00022 TABLE 13 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 22
Anti-Viral Activity of Surgihoney
[0501] S1 or S2 Surgihoney was mixed with Herpes Simplex Virus in cell culture medium (a 50% mixture of honey and virus in cell culture medium) and then incubated for 1 hour at 37 C. The mixtures were then plated onto cells, and the number of viral plaques formed for each mixture was recorded. Controls with no honey, or with control honey were also performed.
[0502] The number of viral (Herpes Simplex Virus) plaques recorded after 1 hour incubation is shown in
[0503] The results show that both the S1 and S2 Surgihoney preparations have potent anti-viral activity.
EXAMPLE 23
[0504] Use of Surgihoney with Line Site Dressings
[0505] To assess the effectiveness of Surgihoney in preventing infection of peripherally inserted central catheters (PICC lines), S1 Surgihoney was applied topically to the line entry site in the arm of 30 patients. Approximately 3 g-8 g S1 Surgihoney was applied to a dressing, which was then contacted with the wound, and held in place by a secondary dressing. Line site colonisation and line-associated bacteraemias were assessed and compared with 30 patients who did not receive the Surgihoney dressing. The results are shown in Table 14 below.
TABLE-US-00023 TABLE 14 Effect of S1 Surgihoney in preventing and clearing line site colonisation Surgihoney Non-Surgihoney (30) (30) Colonised at initiation 2 4 Colonisation during evaluation 0 6 Colonisation cleared during 2 0 evaluation
[0506] It was concluded that Surgihoney is an effective antimicrobial agent for use with line site dressings.
EXAMPLE 24
Use of Surgihoney to Prevent Infection of Caesarean Wounds
[0507] Infection of surgical wounds is a particular problem with Caesarean sections, which have quite a high infection rate of around 10%. There has been a national increase in Caesarean wound infection (8-24.6%) and a wide variation across NHS hospitals (13.6-31.9%) associated with the 147,726 cases of CS each year in the UK (Bragg et al., 2010. Variation in rates of caesarean section among English NHS trusts after accounting for maternal and clinical risk: cross sectional study. BMJ 2010; 341). Caesarean wound infection is a major cause of prolonged hospital stay, resource consumption, as well as other morbidities and mortality. Recovery from Caesarean section is more difficult for women who develop postoperative wound Infection.
[0508] To assess the effectiveness of Surgihoney in preventing infection of Caesarean wounds, S1 Surgihoney was applied once topically to the wound post surgery. Approximately 25 g-35 g S1 Surgihoney was applied to a dressing, which was then contacted with the wound, and held in place by a secondary dressing. Nearly 200 patients were assessed over a three month period.
Clinical Evaluation
[0509] Women presenting for Caesarean section (CS) between October 2012 and January 2013 were offered Surgihoney as a dressing to the wound as a single application when the wound was dressed at the end of the procedure. Each 10 g sachet of Surgihoney was for single patient use. Using an aseptic technique a non-sterile operative assistant opened the Surgihoney sachet and carefully applied the sterile contents on to the sterile dressing. The dressing was then applied to the surgical wound by the obstetrician or theatre midwife. After the procedure, the attending midwife completed an evaluation record. Data collected were MRSA status, history of diabetes, medications, and body mass index. For 14 days after the procedure the attending midwife also recorded any wound healing problems, specifically the presence of oozing, pain, inflammation. If there was any inflammation, a wound culture swab was requested and microbiological results were recorded. The surgical site infection (SSI) rate during the three months of the evaluation using Surgihoney dressing was compared with the infection rate in the 9 months prior to the evaluation based on data collected by the infection control team. Wound infection was defined clinically as an inflamed wound (erythema, swelling, discharge) which required antibiotic treatment. The rate of SSI was calculated as a percentage of all CS procedures carried out.
Results
[0510] The results are shown in Table 15 below. In the 3 month period, October 2012-January 2013, there were 186 CS's, of which 102 (55%) were emergencies. No women were colonised with MRSA. Four (2.23%) had diabetes mellitus. 42 (27.3%) had a body mass index >25. There were 4 out of 186 confirmed CS SSI during the evaluation. This represented an infection rate of 2.15%. A single patient reported an adverse event related to Surgihoney treatment in the form of wound irritation which resolved without further intervention in 3 days. In the preceding 9 months there were 590 CS procedures (234 elective and 356 emergency) and the infection control surveillance recorded 32 CS SSI, representing an infection rate of 5.42%. The reduction in Infection rates is significant: p=0.042 (.sup.2 test).
TABLE-US-00024 TABLE 15 Effect of S1 Surgihoney in preventing infection of Caesarean wounds Total no of Emer- No. of Infection proce- Elective gency infected rate Period dures (%) (%) wounds % January 2012 to 590 234 356 32 5.42% * September 2012 - (39.7%) (60.3%) no S1 Surgihoney 22 Oct. 2012 186 84 102 4 2.15% January 2013 - (45.2%) (54.8%) (60% with S1 Surgihoney reduction) * From Microbiology sample data which probably under reports historic infection rates in the Trust. UK national average closer to 10%
[0511] The results show that there was a low rate of surgical site Infection (a 60% reduction) in the group treated with S1 Surgihoney compared to historic data. The Surgihoney dressing was well tolerated with few reported adverse effects.
[0512] The wound infection rates fell by 60.33% when Surgihoney was used. Using the SSI data from the two arms of the study CS SSI rates (expected) were 5.42% before Surgihoney and (observed) 2.15% after. At these levels (which are lower than the rates of infection previously reported at 9.6%) the extrapolated CS SSI infections rates for UK would be (expected) 8007 cases per year and (observed) 3176 case per year. The difference is 4831 cases that could potentially be reduced by using Surgihoney.
[0513] It was concluded that S1 Surgihoney effectively reduces the rate of infection of Caesarean wounds post surgery. Prevention of colonisation of wounds with Surgihoney, an agent which is not toxic to healing tissue and which also promotes the healing process, is a novel and potentially important finding which may change the way that surgical wounds are managed. Surgihoney offers a clinically and cost-effective intervention to significantly reduce SSI in women undergoing Caesarean section.
Discussion
[0514] This evaluation demonstrated that Surgihoney, a highly effective antimicrobial wound dressing, can be employed as a wound dressing of primary CS wounds to prevent infection. As a natural product with established wound healing properties Surgihoney is likely to promote wound healing in addition to providing potent antimicrobial activity to prevent wound colonisation and infection. Some halogen-based chemical antiseptics may provide the same degree of antimicrobial activity but may delay wound healing (Jan W A. Comparison of conventional pyodine dressing with honey dressing for the treatment of diabetic foot ulcers. JPMIJournal of Postgraduate Medical Institute 2012; 26(4): 402-7). Iodine wound dressings are contraindicated in CS (Joint Formulary Committee. The British National Formulary. London: The Pharmaceutical Press; 2013) and a range of toxicities are associated with their use (Pietsch & Meakins: Complications of povidone-Iodine absorption in topically treated burn patients. The Lancet 1976; 307(7954): 280-2; Scoggin et al.: Hypematrmia and acidosis in association with topical treatment of burns. The Lancet 1977; 309(8018): 959; Donovan et al.: Seizures in a Patient Treated with Continuous Povidone-Iodine Mediastinal Irrigation. New England Journal of Medicine 1992; 326(26): 1784; Colpaert Iodine toxicity as a cause of total atrioventricular block in burn patients. Burns 2009; 35: S45-S6; Ramaswamykanive: Cardiovascular collapse following povidone-lone wash. Anaesthesia and Intensive Care 2011; 39(1): 127-30; Lakhal: Povidone iodine: Features of critical systemic absorption. Armies Francaises d'Anesthesie et de Reanimation 2011; 30(7-8): e1-8):e1-e3).
[0515] Similarly, Cochrane systematic reviews showed there was insufficient evidence to establish whether saver-containing dressings or topical agents promote wound healing, prevent wound infection (Storm-Versloot et al.: Topical silver for preventing wound infection. Cochrane Database of Systematic Reviews 2010) or are effective treatments of infected or contaminated chronic wounds (Vemneulen et al.: Topical silver for treating infected wounds (Review). Cochrane review 2010; (10): 42).
[0516] Although previous systematic reviews on the clinical effectiveness of honey as a wound dressing have shown equivocal evidence of benefit, this new preparation appears to offer significant clinical benefits in CS patients (Jull et al.: Honey as a topical treatment for wounds: The Cochrane Collaboration, 2009; Jull at al.: Honey as a topical treatment for wounds. Cochrane database of systematic reviews (Online) 2013; 2). In a temporal comparison of wound infection rates the evaluation has shown a 60.33% reduction in infection rates from 5.42% prior to the intervention to 2.15% using Surgihoney.
[0517] Healthcare associated infections are a significant and costly healthcare complication with approximately 8% of patients in hospital and SSTs accounted for 14% of these infections and nearly 5% of patients who had undergone a surgical procedure were found to have developed an SSI. SSTs are associated with considerable morbidity and over a third of postoperative deaths are related, at least in part, to SSI. Antimicrobial prophylaxis is routinely employed in many surgical procedures to reduce surgical wound infection. While skin disinfection is also routinely used by surgeons to reduce the skin bacterial load prior to skin incision, it has not been routine practice to use antimicrobial dressings. A reason for this may be that most topical antiseptics have a deleterious effect on tissue healing.
[0518] Surgihoney is a product with potent antimicrobial activity, which is non-toxic and promotes tissue healing. Application of this product topically to dean surgical wounds could actually replace systemic antibiotic prophylaxis in certain types of surgery. Such an advance would assist the reduction of antibiotic volume use and the selection pressure on colonising bacteria.
[0519] Caesarean wounds were chosen in this evaluation because the patients are by and large healthy with no, or very few co-morbidities, and CS Infection rates are reported to be increasing. Possible reasons for this increase have been an increase in older mothers, mothers with co-morbidities, particularly diabetes and a general increase in mothers with higher body mass index. While it has not previously been routine to use an antimicrobial agent in the primary wound dressing, this evaluation has shown an interesting and effective role for Surgihoney in the prevention of CS wound infections.
EXAMPLE 25
Use of S1 Surgihoney to Treat a Pressure Sore
[0520] The patient was a 50 year old female patient with spina bifida who was disabled and immobile. The patient had a pressure sore in the lower beck down to the sacral bone which had persisted for over 1 year. The cavity was infected with Streptococcus pyogenes.
[0521] S1 Surgihoney was used as a topical dressing. Wound improvement was reported from day 2. By day 30, the soft tissue cavity had almost completely healed. No Streptococcus was detected at this point.
[0522] Photographs of the results are shown in
EXAMPLE 26
[0523] Antimicrobial activity of Surgihoney
[0524] 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.
[0525] 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.
[0526] 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.
[0527] Conclusions: Surgihoney is a clinically available wound antiseptic dressing that 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
[0528] 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 % phenol. Values were calculated of the mean from three sample replicates tested, repeated on three days.
Assay Method.
[0529] 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].
Inoculum Preparation.
[0530] 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.
Assay Plate Preparation.
[0531] 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.
Catalase Solution.
[0532] A 200 mg/ml solution of catalase from bovine liver (Sigma C9322, 2900 units/mg) In distilled water was prepared fresh each day.
Sample Preparation.
[0533] 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.
Preparation of Phenol Standards.
[0534] 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.
Sample and Standard Application.
[0535] All samples and standards were tested in triplicate by adding 250 l to each of 3 wells.
Plate Incubation.
[0536] 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.
Calculation of Antibacterial Activity of Samples.
[0537] 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 4.89 and the activity of the samples was then expressed as the equivalent phenol concentration (% w/v).
[0538] Total Activity: all the activity, including activity due to hydrogen peroxide (H.sub.2O.sub.2).
[0539] 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
[0540] The activity was measured using the Merckoquant1.10011. & 1.10081.
Peroxide Test Kits.
[0541] Concentrations expressed as the equivalent mg/L H.sub.2O.sub.2.
[0542] 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.
Method of Determination.
[0543] 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
[0544] 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 Table 16.
2. Determination of Honey Activity by H.sub.2O.sub.2 Method
[0545] 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
Discussion
[0546] 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.
[0547] This peroxide activity offers potent antimicrobial activity that Is ideally suited for a wound dressing that is applied to acute or chronic wounds to treat or prevent wound infections. Whilst a small amount of catalase is present in wounds and serum level of catalase in males has been reported as 50 kU/l it has been shown that catalase activity in healing wounds actually decrease during the first week post-wounding and activity levels of catalase recover to Its original level at two weeks post-wounding. Such concentrations of catalase are thus extremely unlikely to influence the antimicrobial activity observed with exogenously applied Surgihoney or the two modified prototypes, PT1 and PT2.
[0548] The ideal characteristics for an antimicrobial wound dressing are: effectiveness, lack of toxicity, ease of use, patient and clinician acceptability and value for money. 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.
[0549] 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 to wounds continuously over time rather than as a large amount at the time of dressing 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 Dells.
[0550] Cleary Surgihoney and the two modified prototypes, PT1 and PT2 are antimicrobial dressings that offer effective hydrogen peroxide release over at least 24 hours.
Conclusions
[0551] 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 offer a dressing that is effective, non-toxic and easy to administer.
[0552] Table 16 showing the peroxide and non-peroxide antibacterial activities of Surgihoney (SH) and two modified prototypes, PT1 and PT2 against Staphylococcus aureus (NCIMB 9518).
TABLE-US-00025 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
[0553] Examples 27-30 below describe determination of the in vitro antibacterial activity of a composition (Surgihoney) against important biofilm forming burn wound pathogens.
[0554] Surgihoney has previously demonstrated highly potent inhibitory and cidal activity against a wide range of planktonic Gram-positive and Gram-negative bacteria in both laboratory tests and clinical practice. Prophylactic application to caesarean wounds has demonstrated eradication of resistant organisms and reduced rates of colonisation and Infection.
[0555] Given the global concerns surrounding antimicrobial resistance, and the challenges posed by biofilms, the in vitro antibacterial activity of the three different formulations of Surgihoney (S1, S2 and S3as described above) against biofilm-forming isolates of Pseudomonas aeruginosa and Acinetobacter baumannii was Investigated to assess whether Surgihoney can i) prevent the formation of a biofilm and ii) eradicate or prevent seeding of a pre-formed biofilm.
[0556] The anti-biofilming properties of Surgihoney formulations S1, S2 and S3 ware investigated and compared against standard Manuka honey using two biofilm assays. These enabled in vitro measurement of the Minimum Blofilm Inhibition Concentration (MBIC), and the Minimum Biofilm Eradication Concentration (MBEC) of SH. Further experiments we performed to compare the anti-biofilming activity of SH to a range of commercial dressings, including one that contains 20% honey (L-Mesitran net).
EXAMPLE 27
[0557] Prevention of Biofilm Formation by Surgihoney Compared with Manuka Honey
[0558] This example describes a first experiment (Experiment 1), which compares the effect of Surgihoney (S1), and Manuka honey (MH), and a second experiment (Experiment 2), which compares the effect of three different strength preparations of Surgihoney (S1, S2, and S3) on biofilm formation by biofilm-producing isolates of Pseudomonas aeruginosa (control strain PA01, and clinical burn wound isolate 1054) and Acinetobacter baumannii (control strain AYE, and UK ACI clone NCTC_13420(C59)).
Methods
[0559] For each neat honey sample, one loopful of honey was placed Into 1000 water in a well of a 96-well microtitre plate. For the diluted honey samples, approximately 2.5 ml honey was placed into a universal with 6 ml of water. 3 ml of this mixture was then serially diluted down to 1:2048. 100 l of each serial dilution was added to a different well of the 96-well plate.
[0560] Overnight cultures of the isolates were diluted in Muller-hinton broth to an OD600 of 0.1. 100 l of the diluted overnight culture was added to each neat or diluted honey sample well. Each positive control wall contained 100 l diluted overnight culture, and 100 l water. Each negative control well contained 200 l broth or 200 l neat honey sample.
[0561] The plate was incubated for 72 hours at 33 C. (wound temperature) to encourage biofilm formation. The plate was then developed using crystal violet dye (which binds to dead and living biofilms), and visualised following solubilisation of the dye using 70% ethanol.
[0562] A spectrophotometer was then used to assess the optical density (OD) of the solubilised dye. The OD reading corresponds to the amount of incorporated crystal violet. Higher OD readings represent dark wells, and greater mass of biofilm. The OD readings were then plotted to make a graph.
Results
[0563] The results are shown in
TABLE-US-00026 MBIC Experiment 1 Experiment 2 Strain/Isolate S1 MH S1 S2 S3 PA01 1:2 Neat.sup.1 1:4* 1:16* 1:32* 1054 1:2 Neat.sup.1 1:8* 1:32* 1:16* AYE 1:4 1:2 1:8* 1:64* 1:64* C59 1:4 1:2 1:8* 1:64* 1:32* .sup.1The activity of Manuka honey against Pseudomonas was sporadic *= Statistically significant (P < 0.005) when student t-test performed to compare growth with honey and positive control.
[0564] The results show that Surgihoney SH1 prevented Pseudomonas biofilm formation when used neat or at 1:2 dilution, and prevented Acinetobacter biofilm formation when used neat or at 1:2 or 1:4 dilution.
[0565] Manuka honey had a sporadic effect on Pseudomonas biofilm production when used neat, but prevented Acinetobacter biofilm formation when used neat or at 1:2 dilution.
[0566] Surgihoney S2 and S3 also prevented Pseudomonas and Acinetobacter biofilm formation, but were able to do so at lower concentrations than S1 Surgihoney.
[0567] There appeared to be little significant difference in the ability of Surgihoney S2 and S3 to inhibit Pseudomonas and Acinetobacter biofilm formation.
[0568] Biofilm formation by Acinetobacter appeared to be more sensitive to Surgihoney and Manuka honey treatment than Pseudomonas biofilm formation.
Conclusions
[0569] It was concluded from these results that Surgihoney is able to prevent biofilm formation of biofilms, and that Surgihoney S2 and S3 were able to prevent biofilm formation at lower concentrations than Surgihoney S1. Each Surgihoney preparation was able to prevent biofilm formation at a lower concentration than Manuka honey.
EXAMPLE 28
[0570] Prevention of Biofilm Formation by Different Strength Preparations of Surgihoney Compared with Manuka Honey
[0571] This example describes the effect of Surgihoney 1 (S1), Surgihoney 2 (S2), Surgihoney 3 (S3) and Manuka honey (MH), on biofilm formation by biofilm-producing Isolates of Pseudomonas aeruginosa (PS_1586] and PS_6749) and Acinetobacter baumannii (ACI_C60 and ACI_19606).
Methods
[0572] Each honey was placed in a 37 C. incubator for 30 minutes. Approximately 3 ml each honey was then placed into a universal, and 3 ml sterile water was added. This mixture was then vortexed vigorously and serially diluted down to 1:4096.
[0573] 100 l of a diluted overnight culture of each Isolate was added to 100 l of diluted honey in a 96-well microtitre plate, and incubated for 72 hours at 33 C. (wound temperature) to encourage biofilm formation.
[0574] The plate was then developed using crystal violet dye, and visualised following solubilisation of the dye using 70% ethanol.
[0575] A spectrophotometer was then used to assess the optical density (OD) of the solubilised dye. The OD reading corresponds to the amount of incorporated crystal violet Higher OD readings represent dark wells, and greater mass of biofilm. The OD readings were then plotted to make a graph.
Results
[0576] The results are shown in
TABLE-US-00027 MBIC Isolate SH1 SH2 SH3 MH PS_1586 1:8 1:64 1:32.sup.1 .sup.1:2.sup.2 PS_6749 1:8 1:512 1:512 .sup.1:16.sup.3 ACI_C60 .sup.1:16.sup.4 .sup.1:128.sup.4 1:16.sup.1 1:4 ACI_19606 1:16 1:64 1:128 1:2 .sup.11:64 dilution not tested .sup.2Enhanced biofilming observed at 1:4 dilution compared to lower dilutions (see below) .sup.3Enhanced biofilming observed at 1:32 dilution compared to lower dilutions (see below) .sup.4Enhanced biofilming observed at 1:8 to 1:32 dilutions compared to lower dilutions (see below)
[0577] The positive controls (POS) show that all the Isolates were able to form a biofilm, and the negative controls (NEG) show that there was no contamination.
[0578] For some of the isolates, enhanced biofilming is observed at certain concentrations, compared with biofilming at higher and lower concentrations, of the Surgihoney or Manuka honey. This effect has been observed occasionally with other biocides. It is believed to be due to the stress of the biocide causing enhanced biofilming.
Conclusions
[0579] It was concluded from these results that each Surgihoney preparation (S1, S2, and S3) was able to prevent each of the isolates tested from forming a biofilm. The S2 and S3 Surgihoney preparations were able to do so at lower concentrations than S1 Surgihoney.
[0580] The optimum Surgihoney preparation for preventing biofilm formation of the isolates tested was S2. A dilution of 1:64 S2 was able to prevent biofilm formation of each isolate tested.
[0581] Each strength preparation of Surgihoney tested (S1, S2, and S3) was generally able to prevent biofilm formation at lower concentrations than Manuka honey.
EXAMPLE 29
Prevention of Ore-Formed Biofilm Seeding by Surgihoney
[0582] This example describes the effect of Surgihoney S1, S2, and S3 preparations, and Manuka honey (MH), on the prevention or reduction of the seeding of pre-formed biofilms produced by two isolates of Acinetobacter baumannii (ACI_AYE and ACI_C59).
Methods
[0583] 200 l of a serially diluted overnight culture of Acinetobacter baumannii (ACI_AYE or ACI_C59) was added to each well of a 96-well microtitre plate. A PCR peg plate was placed on top of the microtitre plate so that each well contains a peg on which a biofilm can form. The 96-well plate was then incubated for 72 hours at 33 C. to encourage biofilms to grow.
[0584] After 72 hours, the pegs were washed and then placed into a further 96-well plate with wells containing the test agent (Surgihoney or Manuka honey), or broth alone (for the controls). After 24 hours, the pegs were washed and then placed into another 96-well plate containing sterile broth for overnight incubation. The OD of the broth was assessed the following day using the spectrophotometer (as described in Examples 27 and 28 above). Turbid broth has a high OD and represents successful seeding of the biofilm.
[0585] Finally, the pegs were subjected to a crystal violet assay (as described in Examples 27 and 28) to prove that biofilms were present on the pegs in the first place.
Results
[0586] The results are shown in
[0587] In the Figures, the y-axis represents the OD of the broth and, therefore, the amount of seeding. As expected, a large amount of biofilm seeding was observed with each positive control, and minimal background turbidity was observed with each negative control. Biofilms were present on all of the pegs. The results allow determination of the Minimum Biofilm Eradication Concentration (MBEC) of Surgihoney.
S1:
[0588] Reduced seeing was observed as low as the 1:16 dilution of S1 for both isolates. Some turbidity was observed at the 1:2 dilution. It is believed that this may have been an artefact due to some of these test wells being in the corner of the plate and, therefore, being more difficult to wash. Some turbidity was also observed at the 1:8 dilution. Again, this is believed to have been an artefact, possibly because these test wells were next to the positive control on the plate.
S2:
[0589] Reduced seeding was observed for the 1:2 to 1:32/1:84 dilutions for both isolates. As for SH1, some turbidity was observed at the 1:2 dilution. This may have been due to some of these test wells being in the corner of the plate and, therefore, being more difficult to wash.
S3:
[0590] Reduced seeding was observed for the 1:2 to 1:32/1:64 dilutions for both isolates. As for S1 and S2, some turbidity was observed at the 1:2 dilution. This may have been due to some of these test wells being in the corner of the plate and, therefore, being more difficult to wash.
MH:
[0591] Reduced seeding was observed for the 1:2 to 1:16/1:32 dilutions.
Conclusions
[0592] The results confirm that Surgihoney prevented or reduced the seeding of pre-formed biofilms. The S2 and S3 preparations of Surgihoney were able to prevent or reduce biofilm seeding at lower concentrations than the S1 preparation, although there appeared to be little difference in potency between the S2 and S3 preparations. The S2 and S3 preparations appeared to be slightly more potent than Manuka honey in reducing biofilm seeding.
EXAMPLE 30
[0593] Prevention of Biofilm Formation by Surgihoney Compared with Commercially Available Wound Dressings
[0594] This example describes the effect of Surgihoney S1, S2, and S3 preparations on prevention of biofilm formation by Pseudomonas aeruginosa (control strain PA01), and Acinetobacter baumannii (control strain AYE), in comparison with deactivated Surgihoney (DE), Manuka honey (MH: Comvita Manukacare 18+), acetic add (AA), and several commercially available wound dressings, and wound creams.
Methods
[0595] Overnight cultures of the isolates were diluted in Muller-hinton broth to an OD600 of 0.1.
[0596] Surgihoney S1, S2, and S3, deactivated Surgihoney (DE), and Manuka honey (MH: Comvita Manukacare 18+), were tested at serial dilutions of 1:2-1:256 (using water as diluent). 1 ml honey was placed in each well with 1 ml diluted isolate culture.
[0597] 1 cm.sup.2 m.sup.2 of each of the following commercially available dressings was added to 1 ml water and 1 ml diluted isolate culture: Mepilex Ag; Urgotul Ag; Acticoat (Ag); Urgotul (no AM agent); Mesitran Net (honey-based dressing); Polymem (no AM agent).
[0598] Commercially available creams Trimovate and Flamazine (used for treatment of skin infections) were also tested. Acetic add was also tested from 5% down to 0.04%.
[0599] The plate was incubated for 72 hours at 33 C. (wound temperature) to encourage biofilm formation. The plate was then developed using crystal violet dye, and visualised following solubilisation of the dye using 70% ethanol.
[0600] A spectrophotometer was then used to assess the optical density (OD) of the solubilised dye. The OD reading corresponds to the amount of incorporated crystal violet. Higher OD readings represent dark wells, and greater mass of biofilm. The OD readings were then plotted to make a graph.
Results
[0601] The results are shown in
[0602] The results show that Acticoat and Mepilex Ag dressings (and to a lesser extent, Urgotel silver) were effective at preventing biofilm formation of both strains, as was the Flamazine cream. The Urgotul and Polymem dressings, and Mesitran net (a commercial honey-based dressing), and the Trimovate cream did not appear to be effective in preventing biofilm formation.
[0603] Acetic acid was effective at preventing biofilm formation down to 0.31% (AYE) and 0.1% (PA01).
[0604] All of the Surgihoneys tested were effective at preventing biofilm formation of both strains at 1:2 and 1:4 dilution, at least, and some at lower concentrations (for example, S2 was effective against AYE at 1:64 dilution).
[0605] The S2 and S3 Surgihoneys were more effective in preventing biofilm formation at lower concentrations than S1 Surgihoney, and each Surgihoney preparation was more effective at lower concentrations than Manuka honey (see, for example, the effect of the 1:8 dilution for each). Manuka honey was effective in preventing biofilm formation down to 1:2 to 1:4 dilution.
[0606] The results also show that the deactivated Surgihoney (DE) was effective in preventing biofilm formation. However, when the DE honey was tested for hydrogen peroxide activity, it was found to retain some hydrogen peroxide activity, and so did not appear to have been fully Inactivated.
Conclusions
[0607] It was concluded from these results that Surgihoney was comparable in preventing biofilm formation to several commercially available wound dressings, and more effective than Mesitran net (a commercial honey-based dressing).
[0608] In the above examples, four isolates of A. baumanii, and three of P. aeruginosa were tested and Surgihoney was able to prevent biofilm formation of all isolates in a dose-dependent manner. Pre-formed biofilms of A. baumannii were additionally exposed to all Surgihoney formulations for 24 hours. Reduced seeding of the biofilms was observed for both strains tested and was again dose-dependent.
[0609] The dressings experiment (Example 30) revealed Surgihoney to be equally or more effective in biofilm prevention than three of the eight commercial creams and dressings tested. Furthermore, in this in vitro test, Surgihoney was more effective than L-Mesitran net at preventing biofilm formation of single isolates of A. baumanii and P. aeruginosa.
[0610] These results show that Surgihoney has potent anti-biofilming activity against key Gram-negative pathogens of burn wounds, and superior activity to the majority of commercial dressings tested. This composition can be used in place of antibiotics, and in an era of increasing antibiotic resistance, help to reduce inappropriate antibiotic use.
EXAMPLE 31
Antimicrobial Activity of Electrospun Wound Dressings Containing Surgihoney
[0611] An aqueous solution containing 4% by weight polyethylene oxide (PEO) was mixed with Surgihoney in a weight ratio of 80% PEO solution to 20% Surgihoney. The composition was electrospun on to an alginate substrate using an Elmarco NanoSpider electrospinning apparatus, at a voltage of 80 kV, a separation distance of 17 cm and an electrode rotation of 10 rpm, to form a Surgihoney dressing.
[0612] A 5% (by weight) aqueous PEO solution (not containing Surgihoney) was also electrospun on to an alginate substrate, using the same electrospinning apparatus and parameters, to form a control dressing.
[0613] In a first test, an overnight culture of Escherichia coli was spread over the surface of three petri dishes containing nutrient agar. Samples of control dressings and Surgihoney dressings (approx. 22 cm) were placed on the surface of the cultures. The control dressing was placed on the left side of each plate and the Surgihoney dressing was applied to the right side of each plate. The plates were incubated overnight for 18 hrs at 37 C.
[0614] In a second test, an overnight culture of Staphylococcus aureus was spread over the surface of three petri dishes containing nutrient agar. Samples of control dressings and Surgihoney dressings (approx. 22 cm) were placed on the surface of the cultures. The control dressing was placed on the left side of each plate and the Surgihoney dressing was applied to the right side of each plate. The plates were incubated overnight for 18 hrs at 37 C.
[0615] In a third test, a Surgihoney dressing and a control dressing were tested for the presence of hydrogen peroxide. The presence of hydrogen peroxide was determined using the Peroxide Test Kits from Merckoquant (code number 1.10011). The dressings we wetted before application of the Peroxide Test Kits.
Results
[0616] The results of the first test are shown in
[0617] The results of the second test are shown in
[0618] The results of the third test are shown in
EXAMPLE 32
Sprayable Compositions
[0619] A composition containing 1% by weight polyethylene oxide (PEO), 79% by weight deionised water and 20% by weight Surgihoney was added to a pump action spray device.
[0620] The spray device was used to spray the composition on to a hydrogen peroxide test strip. The test strip indicated the presence of hydrogen peroxide in the composition.
[0621] After five months, the spray was re-tested for its ability to generate hydrogen peroxide, by spraying on to a hydrogen peroxide test strip. The test strip indicated the presence of hydrogen peroxide in the composition.
EXAMPLE 33
Anti-Viral Activity of Surgihoney
[0622] 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.4) 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-00028 Experiment 1 Experiment 2 well well well well well well Honey Dilution 1 2 3 1 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
[0623] The results show that SH1 and SH2 Surgihoney was strongly virucidal against HSV in both experiments.
EXAMPLE 34
[0624] Cytotoxic activity of Surgihoney
[0625] SH1 or SH2 Surgihoney (50 g honey diluted 10.sup.2, 10.sup.4, 10.sup.4, 10.sup.4) 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
[0626] The results show that SH1 Surgihoney was cytotoxic at the 10.sup.4 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.
[0627] It is concluded from the results in Examples 33 and 34 that Surgihoney can be administered at doses which are virucidal but not cytotoxic or cytostatic.
TABLE-US-00029 Number of live cells Total number of cells Percentage viability Condi- standard standard standard tion 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 35
[0628] Dried Surgihoney
[0629] Dried honey granules (K24289) were supplied by Kanegrade Limited (Stevenage, UK). The dried honey granules were produced by vacuum drying and contained honey solids with skimmed milk powder. The water content was less than 3%.
[0630] The dried honey granules were activated by adding glucose oxidase at S1 (SH1) and S2 (SH2) levels (see Example 20).
[0631]
[0632]
[0633]
EXAMPLE 36
Stability of Aqueous Surgihoney Compositions
[0634] A sample was prepared with the following composition, on 9 Nov. 2014:
SH2 Surgihoney: 20% by weight
PVA: 5% by weight
Water: 75% by weight
[0635] The sample was tested on 16 Oct. 2015 using a hydrogen peroxide test strip and was found to still be producing hydrogen peroxide at a level of between 30-50 ppm.
[0636] A sample was prepared by mixing SH2 Surgihoney with water in a ratio of 1:15 (Surgihoney:water, by weight), on 14 Sep. 2015. The sample was tested on 16 Oct. 2015 using a hydrogen peroxide test strip and was found to still be producing hydrogen peroxide at a level of 30-50 ppm.
EXAMPLE 37
Use of Surgihoney for the Topical Treatment of Lower Genital Tract Infections
[0637] Surgihoney has been used to treat persistent vaginal discharge that had not been responsive to standard therapy. The indications were vaginal discharge with various aetiologies e.g. bacterial vaginosis and general bacterial vaginal discharge.
[0638] Tampons coated with Surgihoney were inserted Into the vagina and were replaced every 24 hours. The therapeutic outcome was good and there were no reported adverse effects.
EXAMPLE 38
Use of Surgihoney to Treat CPC
[0639] A patient developed a soft tissue infection of the foot whilst in India and was diagnosed with necrotising fascitis. This required extensive debridement of dead tissue as well as antibiotics. Post-operatively, the patient was found to be colonised in the debrided wounds with CPE (Carbapenernase-producing Enterobacteriaceae). The patient required isolation and was treated with Surgihoney, which cleared the organisms.
EXAMPLE 39
Compositions Comprising Non-Aqueous Solvent
Sample 1
[0640] Lightweight hydro entangled fabric (40 g per square meter)
Coated with 25% w/w honey/glycerol solution
Weight of coating on fabric 100 g per square meter
Suggested application: Absorbent fabric in a wound dressing.
Sample 2
[0641] Lightweight hydro entangled fabric (40 g per square meter)
Coated with 75% w/w honey/glycerol solution
Weight of coating on fabric 100 g per square meter
Suggested application: Absorbent fabric in a wound dressing.
Sample 3
[0642] Lightweight hydro entangled fabric (40 g per square meter)
Coated with 25% w/w honey/glycerol solution
Weight of coating on fabric 300 g per square meter
Suggested application: Anti bacterial wipe
Sample 4
[0643] Lightweight hydro entangled fabric (40 g per square meter)
Coated with 75% w/w honey/glycerol solution
Weight of coating on fabric 300 g per square meter
Suggested application: Anti bacterial wipe
Sample 5
[0644] Paper like fabric (42 g per square meter)
Coated with 25% w/w honey/glycerol solution
Weight of coating on fabric 100 g per square meter
Suggested application: And bacterial wipe
Sample 6
[0645] Boots Wound Dressing coated with 25% w/w honey/glycerol solution
[0646] Weight of coating on fabric 400 g per square meter
Sample 7
[0647] Boots Adhesive Wound Dressing with absorbent pad coated with 25% w/w honey/glycerol solution
[0648] Weight of coating on fabric 150 g per sure meter
Sample 8
[0649] Lightweight hydro entangled fabric (40 g per square meter)
Coated with 25% w/w Surgihoney/glycerol solution
Weight of coating on fabric 100 g per square meter
Suggested application: Absorbent fabric in a wound dressing.
Sample 9
[0650] Lightweight hydro entangled fabric (40 g per square meter)
Coated with 25% W/w Surgihoney/glycerol solution
Weight of coating on fabric 300 g per square meter.
EXAMPLE 40
Compositions Comprising Honey, Non-Aqueous Solvent and Additional Water.
[0651] The following composition has been formulated which readily sprays from a Boots Travel Spray bottle (all amounts by weight):
Honey (Surgihoney): 25%
Glycerol: 52.5%
Water: 22.5%
[0652] If the water content in the honey is included, this composition has a mole fraction of water of 68.9%.
[0653] The spray bottle is operated by a manual pump.
Pump inlet tube length: 100 mm
Tube bore: 1.5 mm
Outlet bore: 2.5 mm
Pump delivery: 0.14 g of water per stroke.
EXAMPLE 41
Cytotoxicity of Surgihoney Against Mast Cells
[0654] Mast cells are sentinel cells located Just underneath the epithelium in tissues with close contact to the external environment, such as the nasal mucosal tissues. They play an important role in recognition of pathogens and modulation of immune responses. This example describes the effects of different concentrations of Surgihoney on cells of the human mast cell line HMC-1.
Methods
A) Cell Culture
[0655] Different concentrations of S1, S2 and S3 preparations of Surgihoney, and the control honey (Acacia), were used: 100 g, 40 g, 10 g, 1 g/L. These were made up using a cell growth medium (IMDM, 10% FBS, 2% Penicillin Streptomycin). 2 million HMC-1 cells were added Into 12 well plates, 2 ml of each concentration was used.
B) Cell Survival Assay
[0656] Cell survival was assessed at 3, 6 and 24 hours by staining a 20 l sample with Trypan blue.
C) Live and Dead Assay
[0657] Live and dead cells were counted using a haemocytometer. The percentage of dead cells was calculated and the data was analysed using GraphPad Prism.
Results
[0658] The results of 3, 6 and 24 hour culture of HMC-1 cells with different concentrations of S1, S2, and S3 Surgihoney are shown in
Conclusions
[0659] These results indicate that it may be beneficial to use Surgihoney (particularly S1, S2, or S3 preparations, preferably S2 or S3 preparations), or other compositions of the invention, at concentrations of less than 100 g/L, preferably 40 g/L or less, 10 g/L or less, or 1 g/L or less, for treatment of microbial Infections in areas where mast cells are present, such as epithelium in tissues with close contact to the external environment, for example nasal mucosal tissues.
[0660] It may also be beneficial to limit contact of Surgihoney, or other compositions of the Invention, with cells for less than 24 hours, preferably less than 6 hours, or less than 3 hours, especially when contacting areas where mast cells are present, such as epithelium in tissues with close contact to the external environment, for example nasal mucosal tissues.
EXAMPLE 42
[0661] Electrospinning was undertaken in the following experiments.
[0662] Experiment 1
Composition
[0663] 20% PCL with 0% Surgihoney (by weight) in acetic acid solvent.
Electrospinning Parameters
[0664] Flow rate: 0.8 ml/min
Applied Voltage: 13 kV
Collecting Distance: 15 cm
Processing Time: 40 min
[0665] Experiment 2
Composition
[0666] 20% PCL with 5% Surgihoney (by weight) in acetic acid solvent.
Electrospinning Parameters
[0667] Flow rate: 0.8 ml/min
Applied Voltage: 17 kV
Collecting Distance: 15 cm
Processing Time: 40 min
[0668] Experiment 3
Composition
[0669] 20% PCL with 11% Surgihoney (by weight) in acetic acid solvent.
Electrospinning Parameters
[0670] Flow rate: 0.8 ml/min
Applied Voltage: 16.6 kV
Collecting Distance: 15 cm
Processing Time: 40 min
[0671] Experiment 4
[0672] 20% PCL with 20% Surgihoney (by weight) in acetic add solvent.
Electrospinning Parameters
[0673] Flow rate: 0.8 ml/min
Applied Voltage: 17.5 kV
Collecting Distance: 15 cm
[0674] Processing lime: 40 min
[0675] Experiment 5
Composition
[0676] 20% PCL with 30% Surgihoney (by weight) In acetic add solvent.
Electrospinning Parameters
[0677] Flow rate: 0.8 ml/min
Applied Voltage: 16.5 kV
Collecting Distance: 15 cm
Processing Time: 40 min
[0678] The experiments resulted in the production of nanofibrous mats.
EXAMPLE 43
[0679] Sample process parameters for electrospinning Surgihoney into meshes
TABLE-US-00030 Honey concentration Voltage Flow rate Distance (wt. %) (kV) (mL/min) (cm) 20 11 0.1 13 30 10.5 0.05 13 35 13.5 0.1 13 40 15 0.2 13 45 17 0.4 13 50 18 0.5 13
Diameter Measurements of Fibers Produced
[0680]
TABLE-US-00031 Honey concentration Maximum Minimum Average (wt. %) (m) (m) (m) Control Group 5.45 0.84 2.46 35 16.52 0.41 3.54 40 7.94 1.29 3.70 45 5.45 0.84 2.46
[0681] The control was in the absence of Surgihoney.
Effectiveness of Meshes Against MRSA
[0682]