ANTIMICROBIAL COMPOSITIONS
20220248689 · 2022-08-11
Assignee
Inventors
- Thomas Patton (Rathrippon Collooney, IE)
- James Brennan (Hazelwood, IE)
- John Reginald Barrett (Templemore, IE)
- Ian Staples (Southmoor, GB)
Cpc classification
A01N65/20
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A01N65/00
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A01N65/00
HUMAN NECESSITIES
A01N59/00
HUMAN NECESSITIES
A61L2300/404
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K38/443
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A01N65/20
HUMAN NECESSITIES
A01N59/00
HUMAN NECESSITIES
A61L15/46
HUMAN NECESSITIES
A61L15/40
HUMAN NECESSITIES
International classification
A01N59/00
HUMAN NECESSITIES
A01N65/00
HUMAN NECESSITIES
A23L29/00
HUMAN NECESSITIES
A61L15/40
HUMAN NECESSITIES
Abstract
Storage-stable compositions for generating antimicrobial activity are described. The compositions comprise an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance, such as a honey, that includes a substrate for the enzyme. In certain embodiments, the enzyme is a purified enzyme. In other embodiments, the substrate lacks catalase activity, and the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance. The storage-stable compositions do not include sufficient free water to allow the enzyme to convert the substrate. Use of the compositions to treat microbial infections and wounds is described, as well as methods for their production.
Claims
1. A storage-stable composition for generating antimicrobial activity, which comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide; and an unrefined natural substance that includes a substrate for the enzyme; wherein the composition does not include sufficient free water to allow the enzyme to convert the substrate.
2. A storage-stable composition for generating antimicrobial activity, which comprises: an enzyme that is able to convert a substrate to release hydrogen peroxide; and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.
3. A composition according to claim 1, wherein the unrefined natural substance lacks catalase activity.
4. A composition according to claim 2 or 3, wherein the substance has been treated to remove or inactivate catalase activity.
5. A composition according to any preceding claim, wherein the substance is an unrefined natural sugar substance.
6. A composition according to claim 5, wherein the sugar substance is a honey.
7. A composition according to claim 5, wherein the sugar substance is a honey that has been treated to remove or inactivate catalase activity.
8. A composition according to claim 5, wherein the sugar substance is a syrup, or an inverted syrup.
9. A composition according to any preceding claim, wherein the substance lacks enzyme activity that is able to convert the substrate to release hydrogen peroxide.
10. A composition according to any preceding claim, wherein the substance is a pasteurised substance.
11. A composition according to claim 6, wherein the honey is an unpasteurised honery, preferably a creamed unpasteurised honey.
12. A composition according to any preceding claim, wherein the enzyme is an oxidoreductase enzyme.
13. A composition according to claim 12, wherein the oxidoreductase enzyme is a glucose oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, choline oxidase, pyruvate oxidase, glycollate oxidase, or amioacid oxidase.
14. A composition according to claim 13, wherein the substrate for the oxidoreductase enzyme is D-glucose, hexose, cholesterol, D-galactose, pyranose, choline, pyruvate, glycollate or aminoacid.
15. A composition according to claim 13, wherein the oxidoreductase enzyme is glucose oxidase and the substrate for the oxidoreductase enzyme is D-glucose.
16. A composition according to claim 15, wherein the composition includes at least one unit of glucose oxidase per gram of the composition.
17. A composition according to claim 16, wherein the composition comprises up to 1500 units of glucose oxidase per gram of the composition.
18. A composition according to claim 16 or 17, wherein the composition comprises more than 15 units of glucose oxidase per gram of the composition.
19. A composition according to any of claims 16 to 18, wherein the composition comprises at least 100 units, and preferably 100-500 units, of glucose oxidase per gram of the composition.
20. A composition according to any of claims 16 to 19, wherein the composition comprises at least 500 units, and preferably 500-1000 units, of glucose oxidase per gram of the composition.
21. A composition according to any preceding claim which is a food standard composition.
22. A composition according to any preceding claim, which is a medical grade or medical device grade composition.
23. A composition according to any preceding claim, wherein each of the components of the composition are natural substances.
24. A composition according to any preceding claim in the form of a powder.
25. An antimicrobial composition which comprises a composition according to any of claims 1 to 24 diluted with sufficient water to allow release of hydrogen peroxide by conversion of the substrate by the enzyme.
26. A composition for generating antimicrobial activity, which comprises: an unrefined natural substance; and a purified enzyme that is able to convert a substrate in the unrefined natural substance to release hydrogen peroxide.
27. A composition for generating antimicrobial activity, which comprises: an unrefined natural substance that lacks catalase activity; and an enzyme that is able to convert a substrate in the unrefined natural substance to release hydrogen peroxide, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance.
28. A composition according to claim 26, wherein the unrefined natural substance lacks catalase activity.
29. A composition according to any of claims 26 to 28, which further comprises hydrogen peroxide.
30. A composition according to any preceding claim, which is a sterile composition.
31. A composition according to claim 30, which has been sterilised by exposure to gamma irradiation.
32. An antimicrobial solution, which comprises a composition according to any of claims 26 to 31.
33. A method of sterilising a composition according to any of claims 1 to 29, which comprises exposing the composition to gamma irradiation, preferably 10-70 kGy, more preferably 25-70 kGy, most preferably 35-70 kGy.
34. A method for producing a storage-stable composition for generating antimicrobial activity, which comprises: contacting a purified enzyme that is able to convert a substrate to release hydrogen peroxide with an unrefined natural substance that includes a substrate for the enzyme, wherein the composition does not include sufficient free water to allow the enzyme to convert the substrate.
35. A method for producing a storage-stable composition for generating antimicrobial activity, which comprises: contacting an enzyme that is able to convert a substrate to release hydrogen peroxide with an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme, wherein the composition does not include sufficient free water to allow the enzyme to convert the substrate.
36. A method according to claim 34, wherein the unrefined natural substance lacks catalase activity.
37. A method according to any of claims 34 to 36, wherein the substance is an unrefined natural sugar substance.
38. A method according to any of claims 34 to 37, wherein the substance has been treated to remove or inactivate catalase activity present in the substance and/or any enzyme activity present in the substance that is able to convert the substrate to release hydrogen peroxide, prior to contacting the substance with the enzyme.
39. A method according to claim 38, which further comprises treating the substance to remove or inactivate catalase activity and/or substrate conversion enzyme activity present in the substance, prior to contacting the substance with the enzyme.
40. A method according to claim 39, wherein the substance is treated by heating the substance to inactivate catalase and/or substrate conversion activity, and then cooling the substance, prior to contacting the substance with the enzyme, to a temperature that allows retention of activity of the enzyme.
41. A method according to any of claims 34 to 40, wherein the substance is a honey.
42. A method according to claim 37, wherein the unrefined natural sugar substance is a syrup or an inverted syrup.
43. A method according to claim 42, which further comprises inverting a substance that comprises sucrose to form the inverted syrup.
44. A method according to any of claims 34 to 43, wherein the enzyme is an oxidoreductase enzyme.
45. A method according to claim 44, wherein the oxidoreductase enzyme is a glucose oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, choline oxidase, pyruvate oxidase, glycollate oxidase, or amioacid oxidase.
46. A method according to claim 45, wherein the substrate for the oxidoreductase enzyme is D-glucose, hexose, cholesterol, D-galactose, pyranose, choline, pyruvate, glycollate or aminoacid.
47. A method according to claim 44, wherein the oxidoreductase enzyme is glucose oxidase and the substrate for the oxidoreductase enzyme is D-glucose.
48. A method according to any of claims 34 to 47 for producing a food standard composition, wherein the substance and the enzyme are of food standard.
49. A method according to any of claims 34 to 48, wherein each of the components of the composition are natural substances.
50. A method for producing a composition for generating antimicrobial activity, which comprises: contacting a purified enzyme that is able to convert a substrate to release hydrogen peroxide with an unrefined natural substance that includes a substrate for the enzyme.
51. A method for producing a composition for generating antimicrobial activity, which comprises: contacting an enzyme that is able to convert a substrate to release hydrogen peroxide with an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme.
52. A method according to claim 50, wherein the unrefined natural substance lacks catalase activity.
53. A method according to any of claims 34 to 52, wherein the enzyme and the unrefined natural substance are in powder form.
54. A method for producing an antimicrobial solution, which comprises diluting a storage-stable antimicrobial composition according to any of claims 1 to 24 with sufficient water to allow the enzyme to convert the substrate in the sugar substance and release hydrogen peroxide.
55. A composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, for use as a medicament.
56. A composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, for prevention, treatment, or amelioration of a microbial infection.
57. Use of a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, in the manufacture of a medicament for the prevention, treatment, or amelioration of a microbial infection.
58. A method of preventing, treating, or ameliorating a microbial infection, which comprises administering a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, to a subject in need of such prevention, treatment or amelioration.
59. A method according to claim 58, which comprises topically administering the composition or solution.
60. A use or method according to any of claims 55 to 59, for treating a human or an animal subject.
61. A method of treating a wound, which comprises administering a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, to a wound site.
62. A composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, for treatment of a wound.
63. Use of a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, in the manufacture of a medicament for treatment of a wound.
64. A method of treating inflammation, which comprises administering a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, to a site of inflammation.
65. A composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, for treatment of inflammation.
66. Use of a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, in the manufacture of a medicament for treatment of inflammation.
67. A method of stimulating tissue growth, which comprises administering a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, to a site in need of such stimulation.
68. A composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, for stimulating tissue growth.
69. Use of a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, in the manufacture of a medicament for stimulating tissue growth.
70. A method of debriding a wound, which comprises administering a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, to a wound in need of debridement.
71. A composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, for debriding a wound.
72. Use of a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, in the manufacture of a medicament for debriding a wound.
73. A method of deodorising a wound, which comprises administering a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, to a wound in need of deodorising.
74. A composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, for deodorising a wound.
75. Use of a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, in the manufacture of a medicament for deodorising a wound.
76. A method according to claim 58, wherein the subject is an insect.
77. A method according to claim 76, wherein the insect is a bee.
78. A method according to claim 77 for prevention, treatment, or amelioration of a Nosema infection.
79. A pharmaceutical composition comprising a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, together with a pharmaceutically acceptable carrier, excipient, or diluent.
80. A pharmaceutical composition according to claim 79, wherein the composition is sterile.
81. A pharmaceutical composition according to claim 79 or 80, which is a medical grade, or medical device grade pharmaceutical composition.
82. A wound dressing which comprises a dressing material for dressing a wound, and a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32.
83. An antimicrobial composition adapted for topical administration to a subject, which comprises a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32.
84. An antimicrobial composition adapted for oral administration to a subject, which comprises a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32.
85. A kit which comprises a composition according to any of claims 1 to 31, or a solution according to claim 32, and instructions for administering the composition.
86. A method for preventing or inhibiting microbial growth, with comprises applying a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, to a surface or region where it is desired to prevent or inhibit such growth.
87. A method according to claim 86, which comprises applying the composition or solution to prevent or inhibit growth of a plant or insect pathogen.
88. A method according to claim 86 or 87, which comprises applying the composition or solution to prevent or inhibit growth of Erwinia.
89. A method according to any of claims 86 to 88, which comprises applying the composition or solution to a plant, a fruit, or a vegetable.
90. A method according to claim 89, wherein the vegetable is a potato.
91. Use of a composition according to any of claims 1 to 31, or an antimicrobial solution according to claim 32, to prevent or inhibit microbial growth.
Description
[0174] Preferred embodiments 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
[0214] This example describes a preferred process for production of a storage-stable composition of the invention, and dilution of the storage-stable composition to release hydrogen peroxide.
[0215] Process for Manufacture of “Activated” Honey
[0216] 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.
[0217] 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.
[0218] 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.
[0219] Dilution of “Activated” Honey
[0220] 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
[0221] 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.
[0222] Well Diffusion Assay
[0223] Staphylococcus aureus (NCIMB 9518) was grown on nutrient agar or in nutrient broth.
[0224] 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. 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.
[0225] Plates were incubated for 16 hrs and zones of inhibition were measured using a dial calliper (+/−0.1 mm). The diameter of zones, including the diameter of the well, were recorded.
[0226] 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.
[0227] Honey
[0228] Honey: Pasteurised Ulmo honey.
[0229] Non-activated honey (i.e. honey to which no glucose oxidase had been added) was used as a control.
[0230] Enzyme Preparation
[0231] 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.
[0232] Detection of Hydroqen Peroxide
[0233] Peroxide test from Merckoquant: Cat. No. 1.10011.0002 Measuring range/colour-scale graduation mg/l H2O2 0.5-2-5-10-25.
[0234] 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 mg/l H.sub.2O.sub.2 or, if necessary, estimate an intermediate value.
[0235] 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.
[0236] Results
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[0238] 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.
[0239] The effect of the activated honey is bactericidal.
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[0241] 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
[0243] 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.
[0244] Well diffusion assays as described in Example 2 were carried out using samples of TINEO honey and different phenol standards.
[0245] 1. 10% Phenol Standard
[0246] 2. 20% Phenol Standard
[0247] 3. 30% Phenol Standard
[0248] 4. TINEO Honey (pasteurized TINEO honey containing no added glucose oxidase)
[0249] 5. TINEO Deact (pasteurized TINEO honey, with further heat deactivation)
[0250] 6. Manuka UMF 25+
[0251] 7. TINEO 20+(pasteurized TINEO honey with added glucose oxidase, 0.005% Biozyme enzyme)
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[0254] 1. 10% Phenol Standard
[0255] 2. 20% Phenol Standard
[0256] 3. 30% Phenol Standard
[0257] 4. TINEO Honey Active 25+ (0.005% enzyme w/w)
[0258] 5. TINEO Honey Active 25+ (0.005% enzyme w/w)
[0259] 6. TINEO Honey Active 40+ (0.05% enzyme w/w)
[0260] 7. Manuka Honey UMF 25+
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EXAMPLE 4
[0262] This example describes the results of tests demonstrating the antimicrobial effect of activated Ulmo and Tineo honey.
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[0266] 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
[0267] 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
[0270] This example describes the results of tests of the stability of activated honeys stored for 60 or 90 days.
[0271] 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.
[0272] 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
[0273] 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.
[0274] 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
[0276] This example shows the results of tests of the antimicrobial activity of various different products on their own, and when mixed with activated honey (i.e. honey with added glucose oxidase). Antimicrobial activity against Staphylococcus aureus was tested in a well diffusion assay carried out as described in Example 2. The different samples tested are listed in Table 2 below, and photographs of the results are shown in
TABLE-US-00002 TABLE 2 Sample Number A B 1 Chesty Cough Syrup Formula +10% Activated Honey powder 2 Cough Syrup with honey +10% Activated Honey powder 3 Branded Nappy cream +10% Activated Honey powder 4 Branded Itch Relief Cream +10% Activated Honey powder 5 Branded hand Cream +10% Activated Honey powder 6 Face cleanser +10% Activated Honey powder 7 Branded smoothing facial +10% Activated Honey powder scrub 8 Barrier Vaseline +10% Activated Honey powder 9 Branded baby nappy cream +10% Activated Honey powder 10 Branded gentle cleanser face +10% Activated Honey powder wash 11 Branded face care exfoliating +10% Activated Honey powder wash 12 Branded facial wash +10% Activated Honey powder 13 Branded nappy care ointment +10% Activated Honey powder 14 Antiseptic wound cream +10% Activated Honey powder 15 Branded hand gel with +10% Activated Honey powder Alovera 16 Branded antiseptic wound +10% Activated Honey powder spray 17 Miel vida dulce y sana +0.015% Glucose oxidase 18 Miel de Abejas +0.015% Glucose oxidase 19 Pure Honey sample jars +0.015% Glucose oxidase 20 Propolis tablets +0.15% Glucose oxidase 21 Propolis and Honey spray +0.015% Glucose oxidase 22 Propolis spray +0.015% Glucose oxidase 23 Propolis dropper +0.015% Glucose oxidase 24 Honey powder +2% Glucose oxidase
[0277] The results clearly show the increased antimicrobial effectiveness of the products when mixed with activated honey.
EXAMPLE 9
[0278] This example shows the results of further tests of the antimicrobial activity of various different products on their own, and when mixed with activated honey (i.e. honey with added glucose oxidase). Antimicrobial activity against Staphylococcus aureus was tested in a well diffusion assay carried out as described in Example 2. The different samples tested are listed below, and photographs of the results are shown in
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[0280] 1. Branded antiseptic cream #1
[0281] 2. Branded antiseptic cream #1+10% Ulmo 40+
[0282] 3. Branded antiseptic cream #2
[0283] 4. Branded antiseptic cream #2+10% Ulmo 40+
[0284] 5. TINEO Honey Active 20+
[0285] 6. Manuka Honey 25+
[0286] 7. Ulmo Honey Active 20+
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[0288] 1. Cough syrup
[0289] 2. Cough syrup+10% Ulmo 40+
[0290] 3. Branded hand cream
[0291] 4. Branded hand cream+10% Ulmo 40+
[0292] 5. TINEO Honey Active 20+
[0293] 6. Manuka Honey 25+
[0294] 7. Ulmo Honey Active 20+
[0295] The results clearly show the increased antimicrobial effectiveness of the products when mixed with activated honey.
EXAMPLE 10
[0296] This example describes sterilisation of a composition of the invention that comprises unpasteurised honey and added purified glucose oxidase.
[0297] 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.
[0298] 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 7.3±0.2) 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.
[0299] One positive result was noted after the full incubation period. Substantiation of 35 kGy as a sterilization dose was accepted.
[0300] 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 11
[0301] This example describes the results of tests demonstrating the antimicrobial effect of a composition of the invention 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).
[0302] Well diffusion assays as described in Example 2 were carried out using samples of Manuka honey UMF 25+, Non Active Honey, and Surgihoney.
[0303] 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 12
[0304] Stability testing of a composition of the invention that comprises unpasteurised honey and added purified glucose oxidase that has been sterilised using gamma irradiation (at a minimum dose of 35 kGy).
[0305] 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.
[0306] 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.).
[0307] The relationship between real time and accelerated ageing is as follows:
TABLE-US-00003 TABLE 3 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
[0308] Testing, Test Intervals and Samples Required
[0309] Samples from each batch were 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-00004 TABLE 4 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
[0310] Test Methods
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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 closed 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.
[0315] 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).
[0316] Results
[0317] Filled Sachet Weiqhts—Accelerated Ageing
[0318] Batch A:
TABLE-US-00005 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 10.3 10.1 10.2 10.3 10.3 10.2 10.3 10.3 10.2
[0319] Batch B:
TABLE-US-00006 TABLE 6 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
[0320] Pressure Test Results—Accelerated Ageing
TABLE-US-00007 TABLE 7 Test interval (weeks) Batch A Batch B 2.6 10/10 10/10
pH Test Results—Accelerated Ageing
[0321]
TABLE-US-00008 TABLE 8 Test interval Batch A Batch B (weeks) pH Temp (° C.) pH Temp (° C.) 2.6 3.8 23.1 3.71 23.0
[0322] Moisture Content Test Results—Accelerate Ageing
[0323] Batch A:
TABLE-US-00009 TABLE 9 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
[0324] Batch B:
TABLE-US-00010 TABLE 10 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
[0325] Colour Test Results—Accelerate Ageing
TABLE-US-00011 TABLE 11 Test interval Pfund scale score (weeks) Batch A Batch B 2.6 90-120 90-120
[0326] Examples 13-35 below describe the results of treatment of wounds using a composition of the invention comprising unpasteurised honey with added glucose oxidase (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 13
[0327] This example describes the results of treatment of an infected toe using Surgihoney. The results are shown in
[0328] 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. [0329] a) Day 0—Wound Profile: wound: 3×2×1 cm; 99% Healthy granulation but 1% green colonisation; surrounding skin excoriated; small amount of yellow pus exudate giving off an odour; [0330] b) Day 5—Wound Profile: Wound improved; Second dressing change with 1 honey sachet applied each time; Metrondiazole & amoxicillin; Also Tea tree oil applied; [0331] c) Day 10—Wound Profile: Wound improved; Green colonisation gone; Wound cleaned, dry skin removed and further sachet of honey applied
EXAMPLE 14
[0332] This example describes the results of treatment of a toe ulcer using Surgihoney. The results are shown in
[0333] 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. [0334] a) Day 0—Wound Profile: Wound: 2×2×0.2 cm; 1% yellow brown slough, rest granulated tissue; Low amount of yellow exudate; [0335] b) Day 7—Wound Profile: Wound improved; Daily application on 0.5 sachets of Surgihoney with dressing change; Flucox; [0336] c) Day 10—Wound Profile: Wound improved further; Flucox stopped.
EXAMPLE 15
[0337] This example describes the results of treatment of toe ulcers using Surgihoney. The results are shown in
[0338] The patient was a 61 year old diabetic female with toe ulcers on both 1st and 2nd toes of left foot. Mild pain. [0339] a) Day 0—Wound Profile: Wound: 0.8×0.8×0.2 cm; 100% Granulitic tissue; Small amount of serous exudates; [0340] b) Day 7—Wound Profile: Wound healed to the extent that it is no longer open; Half a sachet of honey each day; Flucox; [0341] c) Day 10—Wound Profile: Wound improved; Now dry, no slough or exudates; Flucox stopped.
EXAMPLE 16
[0342] This example describes the results of treatment of a foot ulcer using Surgihoney. The results are shown in
[0343] 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. [0344] a) Day 0—Wound Profile: Wound: 1×0.5×0.5 cm; 1% yellow brown slough, rest granulated tissue; Very low amount of low, yellow exudates; [0345] b) Day 7—Wound 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; Nil antibiotics; [0346] c) Day 9—Wound Profile: Wound much improved; Nearly closed with no exudates present; Dressings continue to be applied with 0.5 sachets on each.
EXAMPLE 17
[0347] This example describes the results of treatment of a foot ulcer using Surgihoney. The results are shown in
[0348] The patient was an 87 year old diabetic male with a peripheral vascular disease with a diabetic foot ulcer that had developed over more than a year. Surrounding skin was fragile though no pain apparent. [0349] a) Day 0—Wound Profile: Wound: 1×1.8 cm; 1% yellow brown slough, rest granulated tissue; Small amount of yellow serous exudate; [0350] b) Day 3—Wound Profile: Wound improved; 1 honey sachet applied with dressing change; No reports of pain; nil antibiotics; [0351] c) Day 6—Wound Profile: Wound improved; Wound bed now 100% granulitic tissue; Further honey sachet applied; Diprosalic on dry skin surrounding ulcer.
EXAMPLE 18
[0352] This example describes the results of treatment of a diabetic foot ulcer using Surgihoney. The results are shown in
[0353] The patient was a male with a diabetic foot ulcer caused by poor quality shoe irritation. The patient had the ulcer less than 1 month prior to treatment. [0354] a) Day 0—Wound Profile: Red surrounding skim with mild pain; 1% slough, 99% granulated; Infection and diabetes; Low volume exudate, serous and yellow; Wound size: 2 cm×2 cm×0.2 cm; [0355] b) Day 7—Wound 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 cm×1.3 cm×0.1.
EXAMPLE 19
[0356] This example describes the results of treatment of a foot ulcer using Surgihoney. The results are shown in
[0357] The patient was a 55 year old male with severe & poorly controlled diabetes. The patient had a deep ulcer at site of the third toe after amputation. [0358] a) Day 0—Wound Profile: Wound: 3×3×3 cm; 1% yellow brown slough; 1% green colonisation; rest granulitic tissue; Medium volume of yellow puss exudates; [0359] b) Day 1—Wound Profile: Wound improved; All slough removed before honey applied (1 sachet); metronidazole; [0360] c) Day 3—Wound Profile: Wound improved; Further sachet applied; Patient to be discharged; Swab: +++ skin flora; nil antibiotics.
EXAMPLE 20
[0361] This example describes the results of treatment of a traumatic leg wound using Surgihoney. The results are shown in
[0362] The patient was a 95 year old female with a traumatic wound to the lower leg that caused moderate pain. [0363] a) Day 0—Wound Profile: Wound: 15×12×1 cm; Mostly granulitic tissue but with 1% of wound bed necrotic and black; Medium amount of haemoserous exudate; [0364] b) Day 3—Wound Profile: Wound static; No further necrosis but wound much the same; 1 sachet of honey applied; Swab: Entrecoccus sp.; nil antibiotics; [0365] c) Day 8—Wound Profile: Wound improved; Lower volume of exudate and reduced necrotic tissue.
EXAMPLE 21
[0366] This example describes the results of treatment of an infected leg wound using Surgihoney. The results are shown in
[0367] The patient was a 91 year old female with an infected wound to the lower leg. Surrounding skin was fragile and mild pain existed. [0368] a) Day 0—Wound Profile: Wound Size: 4.5×2.5 cm; Mostly red granulitic tissue with 1% yellow/brown slough; Medium volume yellow serous exudate. [0369] b) Day 15—Wound Profile: Wound improved; Size: 4×2 cm; Less slough and serous exudate present; [0370] c) Day 19—Wound Profile: Wound improved; Surgihoney applied with each dressing.
EXAMPLE 22
[0371] This example describes the results of treatment of a foot ulcer using Surgihoney. The results are shown in
[0372] The patient was a 53 year old female diabetic with a chronic diabetic foot ulcer sustained after amputation of 1st, 2nd, 3rd & 4th toes. [0373] a) Day 0—Wound Profile: Wound: 6.5×3×0.2 cm; Granulitic tissue with low amount of yellow serous exudates; [0374] b) Day 5—Wound Profile: Wound size improved slightly but bed condition fairly static; Loose granulation tissue removed; Sachet of honey applied with absorbable dressing; Nil antibiotics; [0375] c) Day 7—Wound Profile: Wound clean with healthy granulation tissue; Dressing changed every 2 days with sachet of honey applied each time; [0376] d) Day 9—Wound Profile: Wound improved; Size reduced and less exudate; Patient to be discharged.
EXAMPLE 23
[0377] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0378] The patient was a female with a Leg Ulcer that had developed over the year before treatment began, and caused mild pain. [0379] a) Day 0—Wound Profile: Healthy surrounding skin; 1% slough, 99% granulated; Medium volume exudate, serous and yellow; Wound size: 7 cm×3 cm; [0380] b) Day 7—Wound Profile: Wound improved and patient in community care; 1 sachet of Surgihoney applied; Size reduced and less exudate present; Pain more bearable.
EXAMPLE 24
[0381] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0382] The patient was a 77 year old male with poor nutritional status and dehydration. Anaemia.ca prostrate. The patient had a leg ulcer that had developed over a 6-12 month period prior to treatment. Mild pain. [0383] a) Day 0—Wound Profile: Wound: 20×10×0.5 cm; 1% yellow brown slough, rest granulated tissue but with medium exudate, pus green; Swab: +++ mixed Coliform & Pseudomonas; Antibiotic: Ben Pen, Clinda; [0384] b) Day 4—Wound Profile: Wound static; Solely honey dressing; Ben Pen & Clinda; [0385] c) Day 8—Wound Profile: Wound improved; All green slough gone; Nil antibiotics; Paracetamol.
EXAMPLE 25
[0386] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0387] The patient was a 91 year old lady with a leg ulcer that has developed over a period of 6 to 12 months. She was hospitalised and in a lot of pain. [0388] a) Day 0—Wound Profile: Pain severe with wound odour; Infection; 10% slough, 50% colonized green, 20% cellulitic, 20% healthy granulation; Wound: 15 cm×5 cm×0.3 cm; Exudate: Low volume, serous and yellow; [0389] b) Day 11—Wound Profile: Pain Mild and wound much improved; 100% healthy granulation; No infection; Wound: 10 cm×5 cm×0.2 cm; Exudate: low, haemoserous, red cm; 50% wound size reduction.
EXAMPLE 26
[0390] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0391] The patient was a 78 year old Male, UTI patient. The patient had an infected leg ulcer. Surrounding skin was macerated. The wound was causing mild pain. [0392] a) Day 0—Wound Profile: Wound size: 5 cm×6 cm×0.5 cm; 98% red granulitic, 1% yellow/brown slough, 1% necrotic tissue; Low volume of haemoserous red exudate; [0393] b) Day 8—Wound Profile: Wound improved; 1 sachet of honey applied each dressing change; Swab reveals scanty skin flora; nil antibiotics.
EXAMPLE 27
[0394] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0395] The patient was an 82 year old female with poor nutritional status and dehydration. The patient had a leg ulcer that had developed over the 3 months prior to treatment. [0396] a) Day 0—Wound Profile: Wound: 3×3×0.5 cm; Wound bed consists of healthy granulitic tissue; Small amount of yellow haemoserous exudate; [0397] b) Day 3—Wound Profile: Wound improved; Swab reveals +++ skin flora; 1 sachet of honey applied with dressing; nil antibiotics; [0398] c) Day 8—Wound Profile: Wound improved; Size of wound reduced and reduced volume of exudate.
EXAMPLE 28
[0399] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0400] The patient was an 83 year old immobile male with low serum albumin. The patient had a leg ulcer that had developed over a 3 month period prior to treatment causing a moderate level of pain. [0401] a) Day 0—Wound Profile: Wound: 20×18×0.5 cm; 1% yellow/brown slough, 1% green colonisation, rest granulated tissue; Medium volume of green serous exudate; Slight wound odour; [0402] b) Day 4—Wound Profile: Wound improved; 2 Surgihoney sachets had been applied; ++ Pseudomonas on swab; No antibiotics given; [0403] c) Day 8—Wound Profile: Wound improved after 2 solely honey dressing changes; +++ Pseudomonas; Significant enough improvement to stop honey.
EXAMPLE 29
[0404] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0405] The patient was a 74 year old male smoker with type 2 respiratory failure and poor mobility. The patient had a septic skin tear that had developed over the week prior to treatment causing moderate pain. [0406] a) Day 0—Wound Profile: Wound: 15×20×0.3 cm; 99% healthy granulation but with 1% critically infected; Medium volume exudate of yellow colouring; Swab reveals scanty Coliforms; Antibiotics: Clindamycin; [0407] b) Day 6—Wound Profile: Wound improved after 2 dressing changes; Solely honey dressing; Ulcer now dry; No growth from swabs; Clindamycin; [0408] c) Day 9—Wound Profile: Wound improved; Still residual cellulitis but ulcers healed; No growth on swabs once again; Clinda.
EXAMPLE 30
[0409] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0410] The patient was an 81 year old male with major extensive ulcers on legs and arms. The patient had peripheral vascular disease and poor nutritional status. The wound had developed over the month prior to treatment. The surrounding skin was excoriated and causing severe pain. [0411] a) Day 0—Wound Profile: Wound: 30×28×1 cm; 1% yellow/brown slough, rest granulated tissue; High volume of red haemoserous exudate; Swab reveals +++ mixed Coliforms & Pseudomonas; [0412] b) Day 6—Wound Profile: Wound improved; scanty Pseudomona; nil antibiotics but oromorph for pain; [0413] c) Day 10—Wound Profile: Wound improved; + Psuedomonas still present; Pain less severe.
EXAMPLE 31
[0414] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0415] The patient was a 67 year old male with peripheral vascular disease. A leg ulcer had developed over the week before treatment. The surrounding skin was fragile and pain moderate. [0416] a) Day 0—Wound Profile: Wound: 1×1×0.2 cm; 1% yellow/brown slough, rest granulated tissue; Small amount of yellow serous exudate in wound; [0417] b) Day 11—Wound Profile: After series of dressing changes where wound was static, now showing signs of improvement; Lower volume of exudate, wound smaller; 1× honey sachet applied with each change; Swab reveals staph aureus++; [0418] c) Day 18—Wound Profile: Wound static; Wound size: 3×3×0.2 cm; staph aureus++ present.
EXAMPLE 32
[0419] This example describes the results of treatment of a leg ulcer using Surgihoney. The results are shown in
[0420] The patient was a 43 old female with large diabetic ulcer that was a month old. The ulcer was complicated with an underlying osteomylelitis, bone debriefed and metal work from calcaneum.
[0421] The patient had broken her foot and the subsequent treatment had lead to bone infection. The lead clinician's opinion was that the patient was very likely to have her foot amputated. [0422] a) Day 0—Wound Profile: Wound with 40% slough, 10% colonised green, 50% healthy granulated; Surrounding skin healthy; Moderate pain; Exudate medium, puss, yellow; Wound 9 cm×8 cm×2 com; No antibiotics; [0423] b) Day 1—Wound Profile: Wound static; Swab: Coliform and Pseudomonas; No antibiotics No analgesic; [0424] c) Day 14—Wound Profile: Wound improved; 1 sachet of Surgihoney used; Wound slowly granulating, much improved; Swab: Scanty skin flora.
EXAMPLE 33
[0425] This example describes the results of treatment of a pressure ulcer using Surgihoney. The results are shown in
[0426] 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. [0427] a) Day 0—Wound Profile: Wound size: 0.7×0.7×0.2 cm; 1% yellow/brown slough, 1%; cellulitic tissue with rest granulitic; Low volume of yellow pus present; [0428] b) Day 3—Wound Profile: Wound healed (closed up); 1 Sachet of Surgihoney applied.
EXAMPLE 34
[0429] This example describes the results of treatment of an infected armpit cut using Surgihoney. The results are shown in
[0430] The patient was a 21 year old female with an infected wound under arm. The surrounding skin was red and mild pain caused. [0431] a) Day 0—Wound Profile: 1% cellulitic, 99% granulated; Low volume exudate with yellow pus; Wound size: 5 cm×5 cm; [0432] b) Day 24—Wound Profile: Wound improved; Honey used over 2 weeks while on holiday; nil antibiotics.
EXAMPLE 35
[0433] This example describes the results of treatment of infection surrounding the entry point of a catheter using Surgihoney. The results are shown in
[0434] 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. [0435] a) Day 0—Wound Profile: Wound: 2 cm×2 cm; 1% cellulitic, rest granulated tissue; Low volume of exudate, red and serous; [0436] b) Day 6—Wound Profile: Wound much improved; No exudates; One sachet of surgihoney applied; Swab taken but no growth; nil antibiotics; [0437] c) Day 14—Wound Profile: Wound improved to the extent that it is no longer an issue.
EXAMPLE 36
[0438] Surqihoney
[0439] Surgihoney is unpasteurised honey with added purified glucose oxidase. Three different preparations of Surgihoney were made with different antimicrobial potencies:
[0440] S1 Surgihoney: 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.
[0441] S2 Surgihoney: unpasteurised honey with 0.1% (w/w) added glucose oxidase. The enzyme used was glucose oxidase (G03B2), 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.
[0442] S3 Surgihoney: unpasteurised honey with 0.25% (w/w) added glucose oxidase. The enzyme used was glucose oxidase (G03B2) from BBI Enzymes Limited, activity 274 Units/mg.
[0443] 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 37
[0444] In Vitro Antimicrobial Activity of Surqihoney
[0445] 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.
[0446] Summary
[0447] Results: Surgihoney demonstrates highly potent inhibitory and cidal activity against a wide range of Gram positive and Gram negative bacteria and fungi. MIC/MBC's Are significantly lower than concentrations likely to be achieved in topical clinical use. 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.
[0448] Conclusions: Surgihoney has wide potential as a highly active topical treatment combining the effects of the healing properties of honey with the potent antimicrobial activity of the bioengineered product 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.
[0449] 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.
[0450] 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.
[0451] 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.
[0452] Methods
[0453] 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.
[0454] 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 p 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 p lactamase producers, 2 of Klebsiella spp., 1 Serratia Marcescens Amp C producer, 4 of Pseudomonas aeruginosa, 1 of Acinetobacter iwoffii, 1 of Propionibacterium acnes, 1 Bacteroides fragilis, and 2 of Candida albicans, 1 of Candida glabrata, 1 of Aspergillus fumigates were tested against Surgihoney.
[0455] Agar Diffusion
[0456] 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.
[0457] 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, Yemani, Sudanese and with medical honey, Medihoney and with antimicrobial dressings containing silver (Silver Aquacell) and iodine (lodoflex). 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 2×2 cm and placed on the surface of the inoculated plates.
[0458] 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 spp., and anaerobically for Propionibacterium sp. And Bacteroides sp.)
[0459] Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations
[0460] 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 μL 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.
[0461] 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.
[0462] All wells including controls were inoculated with 75 μL 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.
[0463] The MIC well and those around the MIC well were sub-cultured on blood agar and incubated at 37° C. for 18 hours to determine the MBC. The MBC was the most dilute concentration which showed no growth after incubation.
[0464] Time Kill Curves
[0465] 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.
[0466] The test and control inocula were kept at 30° C. to simulate the temperature of a superficial skin lesion. Colony counts were performed as above in triplicate at time 0.5, 2, 4, 24, 48, 72 and 168 hours.
[0467] 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.
[0468] Results
[0469] Inhibitory Zone Sizes.
[0470] 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).
[0471] 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 12. 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 12.
[0472] MIC's & MBC's
[0473] Surgihoney demonstrated significant antimicrobial activity against all the isolates tested. MIC's and MBC's were very consistent amongst isolates of the same species whether the isolates were multidrug resistant or highly sensitive. Table 13 lists the MIC and MBC values for isolate species tested by dilution ratio and Table 14 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.
[0474] 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.
[0475] Time Kill Curves.
[0476] 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 (
[0477] Discussion
[0478] 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.
[0479] 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.
[0480] 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.
[0481] 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.
[0482] 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.
[0483] 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.
[0484] 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.
[0485] Conclusion
[0486] 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-00012 TABLE 12 Inhibitory zones sizes with different potencies of Surgihoney (S1, S2, S3) No. of S1 Mean zone S2 Mean zone S3 Mean zone Bacteria strains (range)/mm (range)/mm (range)/mm Methicillin-sensitive 12 36.2 (32-38) 53.4 (44-58) 66.5 (60-72) Staphylococcus aureus (MSSA) Methicillin-resistant 6 35.6 (31-38) 52.6 (48-59) 67.3 (59-73) Staphylococcus aureus (MRSA) Streptococci Beta 6 40.0 (35-42) 44.5 (38-51) 59.2 (53-69) haemolytic Enterococcus spp 5 38.0 (34-39) 49.5 (44-55) 61.8 (59-64) Escherichia coli 6 33.4 (30-37) 49.5 (36-55) 62.7 (59-69) Klebsiella sp. 2 34.2 (30-38) 40.0 (38-42) 57.0 (52-62) Pseudomonas 4 25.8 (20-28) 34.8 (30-38) 50.2 (46-51) aeruginosa Acinetobacter lwoffii 1 32.1 43.7 55.2 Bacteroides fragilis 1 22.3 28.7 34.2 Propionibacterium 1 19.7 23.4 31.9 acnes Candida sp. 2 .sup. 9 (8-10) 15 (15) .sup. 26 (24-28) Aspergillus fumigatus 1 8 12 18
TABLE-US-00013 TABLE 13 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 Growth 1:16 1:16 1:64 1:64 at neat at neat
TABLE-US-00014 TABLE 14 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 Growth 15.6 15.6 3.9 3.9 at neat at neat
EXAMPLE 38
[0487] Anti-Viral Activity of Surqihoney
[0488] 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.
[0489] The number of viral (Herpes Simplex Virus) plaques recorded after 1 hour incubation is shown in
[0490] The results show that both the S1 and S2 Surgihoney preparations have potent anti-viral activity.
EXAMPLE 39
[0491] Use of Surqihoney with Line Site Dressings
[0492] 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 15 below.
TABLE-US-00015 TABLE 15 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
[0493] It was concluded that Surgihoney is an effective antimicrobial agent for use with line site dressings.
EXAMPLE 40
[0494] Use of Surqihoney to Prevent Infection of Caesarean Wounds
[0495] 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.
[0496] 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.
[0497] Clinical Evaluation
[0498] 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.
[0499] Results
[0500] The results are shown in Table 16 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 (72 test).
TABLE-US-00016 TABLE 16 Effect of S1 Surgihoney in preventing infection of Caesarean wounds No. of Total no of Elective Emergency infected Infection rate Period procedures (%) (%) wounds % January 2012 to 590 234 356 32 5.42% * September 2012 - no (39.7%) (60.3%) S1 Surgihoney 22 Oct. 2012 186 84 102 4 2.15% January 2013 - with (45.2%) (54.8%) (60% reduction) S1 Surgihoney * From Microbiology sample data which probably under reports historic infection rates in the Trust. UK national average closer to 10%
[0501] The results show that there was a lower 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.
[0502] 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.
[0503] 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.
[0504] Discussion
[0505] 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. JPMI—Journal 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.: Hypernatromia 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-iodine wash. Anaesthesia and Intensive Care 2011; 39(1): 127-30; Lakhal: Povidone iodine: Features of critical systemic absorption. Annales Francaises d'Anesthesie et de Reanimation 2011; 30(7-8): e1-8):e1-e3.).
[0506] Similarly, Cochrane systematic reviews showed there was insufficient evidence to establish whether silver-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 (Vermeulen et al.: Topical silver for treating infected wounds (Review). Cochrane review 2010; (10): 42).
[0507] 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 et 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.
[0508] Healthcare associated infections are a significant and costly healthcare complication with approximately 8% of patients in hospital and SSIs accounted for 14% of these infections and nearly 5% of patients who had undergone a surgical procedure were found to have developed an SSI. SSIs 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.
[0509] Surgihoney is a product with potent antimicrobial activity, which is non-toxic and promotes tissue healing. Application of this product topically to ‘clean’ 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.
[0510] 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 41
[0511] Use of S1 Surqihoney to Treat an Infected Leg Ulcer
[0512] The patent was a 52 year old male with diabetes and chronic kidney disease. The ulcer was extensive, painful, smelly and generating a high volume of serious exudates.
[0513] S1 Surgihoney was used as a topical dressing alongside antibiotics. By day 7, wound odour had reduced, pain was removed, and a high volume of serious exudate and Pseudomonas infection had been eliminated. The patient was discharged at day 7.
[0514] Photographs of the results are shown in
EXAMPLE 42
[0515] Use of S1 Surqihoney to Treat a Pressure Sore
[0516] 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 back down to the sacral bone which had persisted for over 1 year. The cavity was infected with Streptococcus pyogenes.
[0517] 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.
[0518] Photographs of the results are shown in
EXAMPLE 43
[0519] Antimicrobial Activity of Surqihoney
[0520] 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.
[0521] 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.
[0522] 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.
[0523] 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.
[0524] Methods
[0525] 1. Determination of Honey Activity by Bioassay Method
[0526] The antibacterial activity of Surgihoney (SH) 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.
[0527] Assay Method. The agar well diffusion method used was adapted from the punch plate assay for inhibitory substances described in the Microbiology Standard Methods Manual for the New Zealand Dairy Industry (1982)[Bee Products Standards Council: Proposed standard for measuring the non peroxide activity of honey. In. New Zealand: Bee Products Standards Council; 1982.].
[0528] Inoculum Preparation. Overnight culture was adjusted to an absorbance of 0.5 measured at 540 nm using sterile nutrient broth as a blank and a diluents and a cuvette with a 1 cm pathway.
[0529] Assay Plate preparation. A volume of 100 μl 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 μl of test material (sample or standard) was placed into each well.
[0530] Catalase solution. A 200 mg/ml solution of catalase from bovine liver (Sigma C9322, 2900 units/mg) in distilled water was prepared fresh each day.
[0531] Sample preparation. Primary sample solutions were prepared by adding 4 g of sample to 4 ml of distilled water in universals and placed at 37° C. for 30 minutes to aid mixing. To prepare secondary solutions, 2 ml of the primary sample solution was added to 2 ml of distilled water in universals and mixed for total activity testing and 2 ml of the primary sample solution was added to 2 ml of catalase solution and mixed for non-peroxide activity.
[0532] Preparation of phenol standards. Standards (w/v) 10%, 30%, 50% phenol were prepared by dissolving phenol in water. Phenol standards were brought to room temperature in the dark before use and were mixed thoroughly before addition to test wells. Each standard was placed in three wells to test in triplicate. Standards were kept at 4° C. with an expiry date of one month.
[0533] Sample and standard application. All samples and standards were tested in triplicate by adding 250 μl to each of 3 wells.
[0534] Plate incubation. After application of samples the plates were incubated for approximately 18 hours at 37° C. The diameter of inhibition zones, including the diameter of the well (7.0 mm), was recorded.
[0535] Calculation of antibacterial activity of samples. The mean diameter of the clear zone around each phenol standard was calculated and squared. A standard graph was plotted of % phenol against the square of the mean diameter of the clear zone. A best-fit straight line was obtained using linear regression and the equation of this line was used to calculate the activity of each diluted honey sample from the square of the mean measurement of the diameter of the clear zone. To allow for the dilution (assuming the density of the Surgihoney to be 1.35 g/ml) this figure was multiplied by a factor of 4.69 and the activity of the samples was then expressed as the equivalent phenol concentration (% w/v).
[0536] Total Activity: all the activity, including activity due to hydrogen peroxide (H.sub.2O.sub.2).
[0537] Non-Peroxide Activity: H.sub.2O.sub.2 is removed by treating samples with catalase enzyme.
[0538] 2. Determination of Honey Activity by H.sub.2O.sub.2 Method
[0539] The activity was measured using the Merckoquant® 1.10011. & 1.10081.
[0540] Peroxide Test Kits. Concentrations expressed as the equivalent mg/L H.sub.2O.sub.2.
[0541] 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.
[0542] Method of Determination. Peroxidase transfers oxygen from the peroxide to an organic redox indicator, which is then converted to a blue coloured oxidation product. The peroxide concentration is measured semi-quantitatively by visual comparison of the reaction zone of the test strip with the fields of a colour scale. The reaction zone of the test strip is immersed into the Surgihoney sample for 1 sec, allowing excess liquid to run off the strip onto an absorbent paper towel and after 15 seconds (Cat. No. 110011), 5 seconds (Cat. No. 110081), after which a determination of the colour formed in the reaction zone more precisely coincided with the colour fields scale.
[0543] Results
[0544] 1. Activity Rating
[0545] 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 17.
[0546] 2. Determination of Honey Activity by H.sub.2O.sub.2 Method
[0547] 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
[0548] Discussion
[0549] The results from this work show that the main antimicrobial activity of Surgihoney (SH) 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 (SH) alone. There is a striking linear relationship between the antimicrobial activity and the maximum output of hydrogen peroxide from the three honey prototypes.
[0550] 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.
[0551] 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.
[0552] 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 cells.
[0553] Clearly Surgihoney and the two modified prototypes, PT1 and PT2 are antimicrobial dressings that offer effective hydrogen peroxide release over at least 24 hours.
[0554] Conclusions
[0555] 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.
[0556] Table 17 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-00017 Total Activity Non-Peroxide Sample Name Batch No. (% phenol) Activity (% phenol) Surgihoney 2015-06-018B 32 0 Surgihoney PT1 HHI4110311 65 7 Surgihoney PT2 HHI14110312 83 10
EXAMPLE 44
[0557] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0558] Alcoholic Gel
TABLE-US-00018 Carbopol 940 0.3% Triethanolamine 0.4% (needed for pH and stability control) Active honey (5+) 65% Ethanol 25.0% water qs
[0559] Usage: for the treatment of acne. Not to be used on broken skin.
[0560] “Active Honey (5+)” is used to indicate that more glucose oxidase should be used than in other formulations without the (5+) designation.
EXAMPLE 45
[0561] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0562] Aqueous Spray
[0563] 25 ml plastic bottle containing:
TABLE-US-00019 Active honey 10 g Triton CF 0.1 g Maltodextrin, or corn Starch 1 g
[0564] Usage: wounds in general, and for application to burns
[0565] Triton CF acts as a surfactant in this composition. Maltodextrin, or corn starch acts as a film former.
EXAMPLE 46
[0566] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0567] Non-Aqueous Spray
TABLE-US-00020 Active honey 70% Proplyene Glycol 30%
[0568] Usage: Wounds in General
EXAMPLE 47
[0569] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0570] Dressing—Plastic Pouch
TABLE-US-00021 Range of Calcium Alginate dressings 10 cm × 10 cm, 20 cm × 20 cm Active honey 10% w/w
[0571] Usage: wound debridement, for relatively heavily exudating wounds. This composition provides a relatively moist environment.
EXAMPLE 48
[0572] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0573] Honey and Analqesic
TABLE-US-00022 Active honey 99% Ibuprofen 1%
[0574] Usage: for all stages of wound management to provide pain management in wound healing
EXAMPLE 49
[0575] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0576] Powder
[0577] Active honey
[0578] Maltodextrin
[0579] Ibuprofen
[0580] Microcrystalline Cellulose (CMC)
[0581] Polyvinyl Pyrrolidone (PVP)
[0582] Preferably ibuprofen is included in the composition at 1-2% w/w. The other components can be varied, for example to achieve different levels of moisture absorption.
[0583] Usage: packaging in foil to prevent water uptake. PVP—provides granulation. CMC—provides a moisture desiccant.
EXAMPLE 50
[0584] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0585] Wound Debridement
[0586] Reconstituted Aqueous solution, comprising Active honey (for example, 5-50% Active honey), optionally also including analgesic, preferably ibuprofen, suitably at 1-2% w/w.
[0587] Usage: for example in a dispenser (such as a pump dispenser), as a wound rinse following debridement to provide an early start to wound healing and pain relief.
EXAMPLE 51
[0588] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0589] Foam Dressing
[0590] Polyurethane backing to absorb exudates
[0591] island of calcium alginate soaked in Active honey
[0592] silicone to stop adhering to wound
[0593] Usage: foam-based dressing to assist in mopping up wound exudates.
EXAMPLE 52
[0594] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0595] Fungal Nail Treatment
[0596] Plaster with Active Honey (5+) in a foam well
[0597] Hydroxypropylcellulose
[0598] Glycerol
[0599] Isopropyl Alcohol
[0600] Citric Acid
[0601] Monohydrate
[0602] Usage: for the treatment of fungal nail infections
EXAMPLE 53
[0603] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0604] Throat Spray
[0605] Honey and glycerol, preferably comprising 5-20% honey
[0606] Usage: sore throats. The formulation could be in a dual dispenser for spraying, or reconstituted with water prior to use, and then sprayed.
EXAMPLE 54
[0607] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0608] Pommade
[0609] Active honey 25%
[0610] White petrolatum
[0611] Light liquid paraffin
[0612] Talc
[0613] Kaolin
[0614] Zinc oxide
[0615] Usage: for the treatment of infected eczema
EXAMPLE 55
[0616] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0617] Lip Balm
TABLE-US-00023 Petrolatum 5594 50% Microcrystalline Wax 9% Cyclomethicone D5 31% Active Honey 10%
[0618] Usage: For Chaffed Lips
EXAMPLE 56
[0619] This example sets out a preferred honey-based composition of the invention that comprises honey and added glucose oxidase (referred to as “Active honey”).
[0620] Cream
TABLE-US-00024 Honey .sup. 15% Carbomer 2.63% Dimethicone 0.13% Disodium Lauryl Sulphosuccinate 0.05% Disodium Edetate 0.13% Glycerol 5.26% Silica Colloidal Hydrated 0.33% Poloxamer 0.26% Sodium Hydroxide 0.41% Purified Water 85.03%
[0621] Usage: for treating acne. Honey is known to be active against Propionibacterium acnes.