ANTIMICROBIAL COMPOSITIONS AND FORMULATIONS RELEASING HYDROGEN PEROXIDE

Abstract

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

Claims

1. A composition for generating antimicrobial activity, which comprises: a lipophilic phase; an aqueous phase; an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme.

2. A composition according to claim 1, which is in the form of colloid or a suspension.

3. A composition according to claim 1 or 2, which is in the form of an emulsion.

4. A composition according to any preceding claim, wherein the lipophilic phase comprises an oil or a wax.

5. A composition according to claim 4, wherein the oil or wax is selected from: Aleurites Moluccana Seed Oil; Grape (Vitis Vinifera) Seed Oil; Almond Oil NF; Hybrid Safflower (Carthamus Tinctorius) Oil; Anhydrous Lanolin USP; Isopropyl Myristate; Apricot Kernel Oil; Isopropyl Palmitate; Avocado (Persea Gratissima) Oil; Jojoba (Buxus Chinensis) Oil; Babassu Oil; Lanolin; Beeswax; Macadamia (Ternifolia) Nut Oil; Borage (Borago Officinalis) Seed Oil; Mangifera Indica (Mango) Seed Butter; Brazil Nut Oil; Mineral Oil; C12-15 Alkyl Benzoate; Myristyl Myristate; Cannabis Sativa Seed Oil; Olive (Olea Europaea) Oil; Canola Oil; Oryza Sativa (Rice Bran) Oil; Caprylic/Capric Triglyceride; Peanut Oil NF; Carrot (Daucus Carota Sativa) Seed Oil; Petrolatum; Castor (Ricinus Communis) Oil; PPG-15 Stearyl Ether; Ceresin; Retinyl Palmitate; Cetearyl Alcohol; Safflower (Carthamus Tinctorius) Oil; Cetyl Alcohol; Sesame (Sesamum Indicum) Oil; Cetyl Esters; Shea Butter (Butyrospermum Parkii); Cetyl Palmitate; Soybean (Glycine Soja) Oil; Coconut Oil; Stearic Acid; Daucus Carota Sativa (Carrot) Root Extract; Stearyl Alcohol; Diisopropyl Adipate; Sunflower (Helianthus Annus) Oil; Dimethicone; Sweet Almond (Prunus Amygdalus Dulcis) Oil; Dog Rose (Rosa Canina) Hips Oil; Theobroma Cacao (Cocoa) Seed Butter; Emu Oil; Tocopherol; Evening Primrose Oil.

6. A composition according to claim 4 or 5, wherein the oil or wax is a beeswax.

7. A composition according to any preceding claim, which further comprises one or more emulsifying agents.

8. A composition according to claim 7, wherein the emulsifying agent has an hydrophilic-lipophilic balance (HLB) value, or the emulsifying agents have a combined HLB value, in the range 3-6 or 8-18.

9. A composition according to claim 8, wherein the emulsifying agent is selected from: Calcium Stearoyl Lactylate; Ceteareth-20; Cetearyl Glucoside; Ceteth-10; Ceteth-2; Ceteth-20; Cocamide MEA; Glyceryl Laurate; Glyceryl Stearate; Glyceryl Stearate (and) PEG-100 Stearate; Glyceryl Stearate SE; Glycol Distearate; Glycol Stearate; Isoceteth-20; Isosteareth-20; Lauramide DEA; Laureth-23; Laureth-4; Lecithin; Linoleamide DEA; Methyl Glucose Sesquistearate; Oleth-10; Oleth-10/Polyoxyl 10 Oleyl Ether NF; Oleth-2; Oleth-20; Oleth-20; PEG-100 Stearate; PEG-20 Almond Glycerides; PEG-20 Methyl Glucose Sesquistearate; PEG-25 Hydrogenated Castor Oil; PEG-30 Dipolyhydroxystearate; PEG-4 Dilaurate; PEG-40 Sorbitan Peroleate; PEG-60 Almond Glycerides; PEG-8 Laurate; PEG-80 Sorbitan Laurate; Polysorbate 20; Polysorbate 60; Polysorbate 80; Polysorbate 85; Sodium Stearoyl Lactylate; Sorbitan Isostearate; Sorbitan Laurate; Sorbitan Oleate; Sorbitan Sesquioleate; Sorbitan Stearate; Sorbitan Stearate (and) Sucrose Cocoate; Sorbitan Trioleate; Stearamide MEA; Steareth-2; Steareth-21.

10. A composition according to any of claims 7 to 9, wherein the emulsifying agent is a lecithin.

11. A composition according to any preceding claim which is formulated for topical application.

12. A composition according to any preceding claim which is in the form of a cream or a lotion.

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

14. A composition according to any preceding claim, wherein the composition is a storage-stable composition that does not include sufficient free water to allow the enzyme to convert the substrate.

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

16. A composition according to any preceding claim, wherein the enzyme is an oxidoreductase enzyme.

17. A composition according to claim 16, wherein the oxidoreductase enzyme is a glucose oxidase.

18. A composition according to any preceding claim, wherein the substance includes a purified substrate for the enzyme.

19. A composition according to claim 18, wherein the purified substrate comprises a purified sugar.

20. A composition according to claim 19, wherein the purified sugar comprises D-glucose, hexose, or D-galactose.

21. A composition according to any of claims 1 to 17, wherein the substance is an unrefined natural substance.

22. A composition according to claim 21, wherein the unrefined natural substance lacks catalase activity.

23. A composition according to claim 21 or 22, wherein the unrefined natural substance is an unrefined natural sugar substance.

24. A composition according to claim 23, wherein the unrefined natural sugar substance is a honey.

25. A composition according to claim 24, wherein the honey is an unpasteurised honey, preferably a creamed unpasteurised honey.

26. A composition according to any preceding claim, which does not include any detectable hydrogen peroxide.

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

28. A composition according to any preceding claim, wherein the ratio of the lipophilic phase to the aqueous phase is from 9:1 to 1:9, 8:1 to 1:8, 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, or 2:1 to 1:2 (v/v).

29. A composition according to any preceding claim, which comprises 5-95%, 10-95%, 15-95%, 20-95%, 25-95%, 30-95%, 35-95%, 40-95%, 45-95%, 50-95%, 55-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95%, 85-95%, or 90-95% (v/v) lipophilic phase (including any emulsifying agent present).

30. A composition according to any of claims 1 to 28, which comprises 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, or 5-10% (v/v) lipophilic phase (including any emulsifying agent present).

31. A composition according to any preceding claim, which comprises 5-95%, 10-95%, 15-95%, 20-95%, 25-95%, 30-95%, 35-95%, 40-95%, 45-95%, 50-95%, 55-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95%, 85-95%, or 90-95% (v/v) aqueous phase.

32. A composition according to any of claims 1 to 31, which comprises 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, or 5-10% (v/v) aqueous phase.

33. A composition according to any preceding claim, which comprises 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, or 1-10% (w/v) of the substance.

34. A composition according to any of claims 1 to 32, which comprises 1-60%, 5-60%, 10-60%, 15-60%, 20-60%, 25-60%, 30-60%, 35-60%, 40-60%, 45-60%, or 50-60% (w/v) of the substance.

35. A composition according to any preceding claim, which comprises less than 20% water, preferably 10-19% water.

36. A composition according to any preceding claim, which comprises 1-1500 units, 15-1500 units, 30-1500 units, 50-1500 units, 100-1500 units, 1-<685 units, 15-<685 units, 30-<685 units, 50-<685 units, 100-<685 units, 500-1000 units, 685-1000 units, or 100-500 units, of the enzyme, preferably glucose oxidase, per gram of the composition.

37. A composition according to any preceding claim, which comprises sufficient enzyme and substrate to provide for sustained release of 0.1 to less than 2 mmol/litre hydrogen peroxide for a period of at least 24 hours.

38. A composition according to any preceding claim for use as a medicament.

39. A composition according to any of claims 1 to 37 for use in the prevention or treatment of a microbial infection.

40. Use of a composition according to any of claims 1 to 37 in the manufacture of a medicament for the prevention or treatment of a microbial infection.

41. Use according to claim 39 or 40, wherein the prevention or treatment is by topical administration of the composition.

42. A method of prevention or treatment of a microbial infection, which comprises administering an effective amount of a composition according to any of claims 1 to 37 to a subject in need of such treatment.

43. A method according to claim 42, wherein the composition is administered topically to the subject.

44. Use according to any of claims 39 to 41, or a method according to claim 42 or 43, wherein the microbial infection is a viral infection, preferably a herpes simplex virus (HSV) infection.

45. Use according to any of claims 39 to 41, or a method according to claim 42 or 43, wherein the microbial infection is a fungal infection.

46. Use according to any of claims 39 to 41, or a method according to claim 42 or 43, wherein the microbial infection is a bacterial infection.

47. A method of making a composition according to any of claims 1 to 37, which comprises mixing a lipophilic component, an aqueous component, an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substance that includes a substrate for the enzyme to form the composition.

48. A composition for generating antimicrobial activity, comprising: a first phase; a second phase; an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme, wherein the first phase and the second phase are immiscible.

49. A composition according to claim 48, wherein the first phase is less polar than the second phase.

50. A composition according to claim 48 or claim 49, wherein the first phase is a non-polar phase, and the second phase is a polar phase.

51. A composition according to any of claims 48 to 50, wherein the first phase is a lipophilic phase and the second phase is an aqueous phase.

52. A composition according to any of claims 48 to 51, wherein the second phase comprises a non-aqueous solvent, optionally glycerol, dimethylsulphoxide, propylene glycol or polyethylene glycol.

53. A composition according to any of claims 48 to 52, wherein the first phase is, or comprises, an oil, optionally olive oil, corn oil, vegetable oil, sunflower oil or paraffin oil.

54. A composition according to any of claims 48 to 53, comprising an emulsifier, preferably a surfactant, optionally wherein the surfactant is, or comprises, TWEEN, SPAN, Poloxamer or Polyglycerol polyricinoleate.

55. A composition for generating antimicrobial activity, comprising: an oil; an emulsifier; an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme.

56. A composition according to claim 55, comprising a non-aqueous solvent.

57. A composition according to claim 56, wherein the non-aqueous solvent is a polar solvent.

58. A composition according to claim 56 or claim 57, wherein the non-aqueous solvent is an organic solvent.

59. A composition according to any of claims 56 to 58, wherein the non-aqueous solvent is, or comprises, glycerol, dimethylsulphoxide, propylene glycol or polyethylene glycol.

60. A composition according to any of claims 55 to 59, wherein the oil is, or comprises, olive oil, corn oil, vegetable oil, sunflower oil or paraffin oil.

61. A composition according to any of claims 55 to 60, wherein the emulsifier is, or comprises, a surfactant.

62. A composition according to claim 61, wherein the surfactant is, or comprises, TWEEN, SPAN, Poloxamer or Polyglycerol polyricinoleate.

63. A composition according to any of claims 48 to 62, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance.

64. A composition according to any of claims 48 to 63, which does not comprise sufficient free water to allow the enzyme to convert the substrate.

65. A composition according to any of claims 48 to 64 which is storage stable.

66. A composition according to any of claims 48 to 65, which provides for sustained release of hydrogen peroxide at a level of less than 2 mmol/litre for a period of at least twenty four hours, following dilution of the composition.

67. A composition according to any of claims 48 to 66, which provides for sustained release of at least 0.1, 0.5, 1 or 1.5 mmol/litre hydrogen peroxide for a period of at least 24 hours.

68. A composition according to any of claims 48 to 67, wherein the enzyme is a purified enzyme.

69. A composition according to any of claims 48 to 68, wherein the enzyme is an oxidoreductase enzyme, preferably glucose oxidase.

70. A composition according to any of claims 48 to 69, wherein the substance lacks catalase activity.

71. A composition according to any of claims 48 to 70, wherein the substance is an unrefined substance, such as an unrefined natural substance.

72. A composition according to any of claims 48 to 71, wherein the substance is a sugar substance, preferably comprising glucose.

73. A composition according to any of claims 48 to 72, wherein the substance is, or comprises, honey.

74. A composition according to any of claims 48 to 70, wherein the substance includes a purified substrate for the enzyme, preferably a purified sugar such as purified glucose.

75. A composition according to any of claims 48 to 74, comprising 10-60%, preferably 20-50%, more preferably 35-45% (w/w) non-aqueous solvent.

76. A composition according to any of claims 48 to 75, comprising 10-40%, preferably 20-30% (w/w) oil.

77. A composition according to any of claims 48 to 76, comprising 1-10%, preferably 1-5% (w/w) emulsifier

78. A composition according to any of claims 48 to 77, comprising 10-50%, preferably, more preferably 20-40% (w/w) substance which comprises a substrate for the enzyme.

79. A composition according to any of claims 48 to 74, comprising 20-50% (w/w) non-aqueous solvent, 20-30% (w/w) oil, 1-5% (w/w) emulsifier and 20-40% (w/w) substance which comprises a substrate for the enzyme.

80. A composition according to any of claims 48 to 74, comprising 10-60% (w/w) non-aqueous solvent, 10-40% (w/w) oil, 1-10% (w/w) emulsifier and 10-50% (w/w) substance which comprises a substrate for the enzyme.

81. A composition according to any of claims 48 to 74, comprising 35-45% (w/w) non-aqueous solvent, 20-30% (w/w) oil, 1-5% (w/w) emulsifier and 25-35% (w/w) substance which comprises a substrate for the enzyme.

82. A composition according to any of claims 48 to 74, wherein the ratio of the first phase to the second phase is 1:1 (v/v).

83. A composition according to claim 82, wherein the ratio of the first phase to the second phase is <0.6:1 (v/v).

84. A composition according to claim 82, wherein the ratio of the first phase to the second phase is 0.4:1 (v/v).

85. A composition according to any of claims 48 to 74, or 82 to 84, wherein the first phase is present at less than 60% (v/v) of the composition.

86. A composition according to claim 85, wherein the first phase is present at 10% to less than 60% (v/v) of the composition.

87. A composition according to claim 85, wherein the first phase is present at 10% to less than 40% (v/v) of the composition.

88. A composition according to claim 85, wherein the first phase is present at 10% to less than 30% (v/v) of the composition.

89. A composition according to claim 85, wherein the first phase is present at 10% to less than 25% (v/v) of the composition.

90. A composition according to any of claims 48 to 74, or 82 to 89, which comprises an emulsifier, wherein the emulsifier is present at up to 25% (v/v) of the composition.

91. A composition according to claim 90, wherein the emulsifier is present at 1-25% (v/v) of the composition.

92. A composition according to claim 90, wherein the emulsifier is present at 5-25% (v/v) of the composition.

93. A composition according to claim 90, wherein the emulsifier is present at 10-25% (v/v) of the composition.

94. A composition according to any of claims 48 to 74, or 82 to 93, wherein the ratio of the amount of the substance that includes a substrate for the enzyme to the volume of the second phase is from 0.5:1 to 2:1.

95. A composition according to claim 94, wherein the ratio of the amount of the substance that includes a substrate for the enzyme to the volume of the second phase is 1:1.

96. A composition according to any of claims 48 to 74, or 82 to 95, wherein the amount of the substance that includes a substrate for the enzyme in the composition is up to 70% (w/v) of the composition.

97. A composition according to claim 96, wherein the amount of the substance that includes a substrate for the enzyme in the composition is 5-70% (w/v) of the composition.

98. A composition according to claim 96, wherein the amount of the substance that includes a substrate for the enzyme in the composition is 10-70% (w/v) of the composition.

99. A composition according to claim 96, wherein the amount of the substance that includes a substrate for the enzyme in the composition is 20-70% (w/v) of the composition.

100. A composition according to claim 96, wherein the amount of the substance that includes a substrate for the enzyme in the composition is 30-70% (w/v) of the composition.

101. A composition according to any of claims 48 to 100, which is an emulsion, preferably wherein the emulsion comprises reverse micelles.

102. A composition according to claim 101, wherein the reverse micelles are formed by the second phase.

103. A composition according to any of claims 48 to 102, wherein the enzyme and the substance that includes a substrate for the enzyme is dissolved in the second phase.

104. A composition according to any of claims 48 to 103, wherein the first phase is, or comprises paraffin oil.

105. A composition according to any of claims 48 to 104, wherein the second phase is, or comprises glycerol.

106. A composition according to any of claims 48 to 105, wherein the emulsifier is, or comprises Polyglycerol polyricinoleate (PGPR).

107. A composition according to any of claims 48 to 106, wherein the enzyme that is able to convert a substrate to release hydrogen peroxide is, or comprises purified glucose oxidase, and the substance that includes a substrate for the enzyme is, or comprises honey.

108. A composition according to any of claims 48 to 106, wherein the enzyme that is able to convert a substrate to release hydrogen peroxide is, or comprises purified glucose oxidase, and the substance that includes a substrate for the enzyme is, or comprises purified glucose.

109. A composition according to any of claims 48 to 108, which is a cream.

110. A composition according to any of claims 48 to 109, which further comprises a viscosity-increasing agent.

111. A composition according to claim 110, wherein the viscosity-increasing agent is, or comprises a hydrocolloid.

112. A composition according to claim 111, wherein the hydrocolloid is, or comprises a polysaccharide.

113. A composition according to claim 111 or 112, wherein the hydrocolloid is, or comprises a hydrocolloid thickener.

114. A composition according to claim 113, wherein the hydrocolloid thickener is starch, modified starch, xanthan, a galactomannan (such as guar gum, locust bean gum, and tara gum), gum Arabic or acacia gum, gum karaya, gum tragacanth, konjac maanan, or a cellulose derivative, such as carboxymethyl cellulose, methyl cellulose, or hydroxypropylmethyl cellulose.

115. A composition according to claim 111 or 112, wherein the hydrocolloid is, or comprises a cross-linked hydrocolloid.

116. A composition according to claim 115, wherein the cross-linked hydrocolloid is a cross-linked polysaccharide.

117. A composition according to claim 116, wherein the cross-linked polysaccharide is cross-linked alginate, pectin, carrageenan, gelatin, gellan, agar, agarose, modified starch, or a cellulose derivative, such as methyl cellulose or hydroxypropylmethyl cellulose.

118. A composition according to claim 115 or 116, wherein molecules of the hydrocolloid are crosslinked by cations.

119. A composition according to claim 118, wherein the hydrocolloid is alginate, carrageenan or pectin.

120. A composition according to claim 119, wherein the hydrocolloid is alginate cross-linked by calcium ions.

121. A method of making a composition, comprising mixing a first component, a second component, an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substance that includes a substrate for the enzyme to form the composition, wherein the first component and second component are immiscible.

122. A method of making a composition, comprising mixing an oil, an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substance that includes a substrate for the enzyme to form the composition.

123. The method according to claim 121 or 122 comprising mixing a non-aqueous solvent.

124. A method of making a composition according to any of claims 48 to 120, which comprises mixing the enzyme, the substance that includes a substrate for the enzyme, liquid of the second phase, liquid of the first phase, and optionally an emulsifier, under a high rate of shear for sufficient time to form an emulsion.

125. A method according to claim 124, wherein the enzyme, the substance that includes a substrate for the enzyme, liquid of the second phase, and liquid of the first phase are pre-mixed under a high rate of shear before contacting the pre-mixed ingredients with the emulsifier and mixing of the mixture comprising the pre-mixed ingredients and the emulsifier under the high rate of shear.

126. A method according to claim 124 or 125, wherein the enzyme and the substance that includes a substrate for the enzyme are dissolved in the liquid of the second phase to form a solution before contacting the solution with the liquid of the first phase.

127. A method according to any of claims 124 to 126, wherein the high rate of shear is 1000 1/s to 4000 1/s.

128. A method according to any of claims 124 to 127, wherein the high rate of shear is >2500 1/s to 3500 1/s.

129. A method according to any of claims 124 to 128, wherein the mixing is carried out at 20 C. to 40 C.

130. A method according to any of claims 124 to 129, wherein the mixing is carried out at 35 C. to 40 C.

131. A method according to any of claims 124 to 130, wherein the mixing is carried out at 38 C. to 40 C.

132. A method according to any of claims 124 to 131, wherein the enzyme, the substance that includes a substrate for the enzyme, liquid of the second phase, liquid of the first phase, and the emulsifier (if present), are mixed under a high rate of shear for at least 5 minutes.

133. A method according to any of claims 124 to 132, wherein the enzyme, the substance that includes a substrate for the enzyme, liquid of the second phase, liquid of the first phase, and the emulsifier (if present), are mixed under a high rate of shear for 5 to 30 minutes.

134. A method according to any of claims 124 to 133, which further comprises mixing a viscosity-increasing agent with the enzyme, the substance that includes a substrate for the enzyme, liquid of the second phase, liquid of the first phase, and the emulsifier (if present), under the high shear rate to form a cream.

135. A method according to claim 134, wherein the viscosity-increasing agent is, or comprises a hydrocolloid.

136. A method according to claim 135, wherein the hydrocolloid is, or comprises a polysaccharide.

137. A method according to claim135 or 136, wherein the hydrocolloid is, or comprises a hydrocolloid thickener.

138. A method according to claim 137, wherein the hydrocolloid thickener is starch, modified starch, xanthan, a galactomannan (such as guar gum, locust bean gum, and tara gum), gum Arabic or acacia gum, gum karaya, gum tragacanth, konjac maanan, or a cellulose derivative, such as carboxymethyl cellulose, methyl cellulose, or hydroxypropylmethyl cellulose.

139. A method according to claim 135 or 136, wherein the hydrocolloid is, or comprises a hydrocolloid gelling agent.

140. A method according to claim 139, wherein the hydrocolloid gelling agent comprises alginate, pectin, carrageenan, gelatin, gellan, agar, agarose, modified starch, or a cellulose derivative, such as methyl cellulose or hydroxypropylmethyl cellulose.

141. A method according to claim 139 or 140, wherein the hydrocolloid gelling agent is capable of forming a gel by ionotropic gelation in the presence of cations, and wherein the method further comprises mixing the cations with the hydrocolloid gelling agent, the enzyme, the substance that includes a substrate for the enzyme, liquid of the second phase, liquid of the first phase, and the emulsifier (if present), under the high shear rate to form the cream.

142. A method according to claim 141, wherein the hydrocolloid gelling agent, the enzyme, the substance that includes a substrate for the enzyme, liquid of the second phase, liquid of the first phase, and the emulsifier (if present), are mixed to form a mixture prior to contacting the cations with the mixture.

143. A method according to claim 142, wherein the cations are provided in non-aqueous solution, such as ethanol or acetic acid.

144. A method according to any of claims 141 to 143, wherein the cations are, or comprise calcium ions.

145. A method according to any of claims 141 to 144, wherein the hydrocolloid gelling agent is contacted with the liquid of the second phase, the enzyme, and the substance that includes a substrate for the enzyme, prior to contact with the liquid of the first phase.

146. A method according to any of claims 141 to 145, wherein the hydrocolloid gelling agent is, or comprises alginate, carrageenan or pectin.

147. A method according to claim 146, wherein the hydrocolloid gelling agent is, or comprises alginate.

148. A method according to any of claims 141 to 147, wherein the hydrocolloid gelling agent is provided in aqueous solution, and the second phase is glycerol, wherein the glycerol is present in sufficient amount to bind free water in the composition and thereby prevent the enzyme catalysing release of hydrogen peroxide from the substance that includes a substrate for the enzyme.

149. A pharmaceutical composition comprising a composition according to any of claims 48 to 120, and a pharmaceutically acceptable carrier excipient or diluent.

150. A composition according to any of claims 48 to 120, or a pharmaceutical composition according to claim 149, for use as a medicament.

151. A composition according to any of claims 48 to 120, or a pharmaceutical composition according to claim 149, for use in the prevention or treatment of microbial infection.

152. Use of a composition according to any of claims 48 to 120, or a pharmaceutical composition according to claim 149, in the manufacture of a medicament for the prevention or treatment of microbial infection.

153. A method of preventing or treating a microbial infection which comprises administering an effective amount of a composition according to any of claims 48 to 120, or a pharmaceutical composition according to claim 149, to a subject in need of such a treatment.

154. A composition according to claim 151, use according to claim 152, or a method according to claim 153, wherein the microbial infection is: a nasal infection, such as sinusitis or rhinosinusitis; a respiratory tract infection, such as an upper respiratory tract infection (e.g. tonsillitis, laryngitis or sinusitis) or a lower respiratory tract infection (e.g. bronchitis, pneumonia, bronchiolitis or tuberculosis); an infection associated with chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, asthma, or an HIV/AIDS-associated respiratory infection, or a respiratory infection associated with terminal disease.

Description

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

[0251] FIG. 1 shows the results of an assay for the cytotoxic activity of Surgihoney;

[0252] FIG. 2A shows different hydrogen peroxide production rates for Surgihoney SH1, SH2, and SH3;

[0253] FIG. 2B shows the relationship between phenol activity and maximum hydrogen peroxide activity in Surgihoney SH1, SH2, and SH3;

[0254] FIG. 3 shows time kill curves for Surgihoney 1 (S1), Surgihoney 3 (S3), and Medihoney (MH) for different test organisms: (a) Staphylococcus aureus; (b) Methicillin-resistant Staphylococcus aureus (MRSA); (c) E. coli; (d) vancomycin resistant enterococcus (VRE); (e) Pseudomonas aeruginosa; (f) Klebsiella; (g) E. coli ESBL; (h) Enterococcus faecalis; and

[0255] FIG. 4 shows an optical microscopy images of reverse micelles in an emulsion containing Surgihoney.

EXAMPLE 1

[0256] Surgihoney Cream Formulation

[0257] A cream formulation comprising Surgihoney SH1 was made with the following ingredients: [0258] beeswax (for the lipophilic phase); [0259] soya lecithin (as an emulsifier); [0260] water; and [0261] SH1 Surgihoney.

[0262] The cream remains stable for many years, as judged by its ability to produce hydrogen peroxide when contacted with water.

EXAMPLE 2

[0263] Anti-Viral Activity of Surgihoney

[0264] SH1 or SH2 Surgihoney was mixed with Herpes Simplex Virus Type 1 or 2 (HSV 1 or HSV 2) in cell culture medium (a 50% mixture of honey and virus in cell culture medium) and then incubated for 1 hour at 37 C. A dilution series was then made from the mixture, and the dilutions were plated onto Vero cells. SH1 Surgihoney reduced the titre of virus by 1 log. SH2 Surgihoney was virucidal for both HSV 1 and HSV 2 (>6 log drop in titre). The experiment was repeatable.

EXAMPLE 3

[0265] Anti-Viral Activity of Surgihoney

[0266] SH1 or SH2 Surgihoney was mixed with Herpes Simplex Virus (HSV) (50 g honey and 50 l virus) and incubated for 1 hour at 37 C. A dilution series (10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5) was then made from the mixture, and the dilutions were used in a plaque reduction assay. Controls with no honey, or with control honey were also performed. The number of viral plaques formed for each dilution was recorded. The results are shown in the Table below.

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

[0267] The results show that SH1 and SH2 Surgihoney was strongly virucidal against HSV in both experiments.

EXAMPLE 4

[0268] Cytotoxic Activity of Surgihoney

[0269] SH1 or SH2 Surgihoney (50 g honey diluted 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5) was incubated on cells for 2 days. The number of live cells, and the total number of cells was counted (percentage viability=live/total100). The results are shown in the table below, and in FIG. 1.

[0270] The results show that SH1 Surgihoney was cytotoxic at the 10.sup.2 dilution, and cytostatic at the 10.sup.3 and 10.sup.4 dilutions, and that SH2 Surgihoney was cytostatic at the 10.sup.2, 10.sup.3 and 10.sup.4 dilutions. SH1 and SH2 Surgihoney were not cytotoxic or cytostatic at the 10.sup.5 dilution.

[0271] It is concluded from the results in Examples 3 and 4 that Surgihoney can be administered at doses which are virucidal but not cytotoxic or cytostatic.

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

EXAMPLE 5

[0272] Antimicrobial Activity of Surgihoney

[0273] 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.

[0274] 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.

[0275] 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.

[0276] Conclusions: Surgihoney shows good antimicrobial activity. Two further honey prototypes have been shown to have antimicrobial activity that is possible to be enhanced due to demonstrated increases in peroxide activity.

[0277] Methods

[0278] 1. Determination of Honey Activity by Bioassay Method

[0279] The antibacterial activity of Surgihoney (S) and two modified honeys, Prototype 1 (PT1) and Prototype 2 (PT2) was measured using Staphylococcus aureus (NCIMB 9518) and expressed as the equivalent percentage phenol. Values were calculated of the mean from three sample replicates tested, repeated on three days.

[0280] 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.].

[0281] 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.

[0282] Assay Plate preparation. A volume of 100 l of the culture adjusted to 0.5 absorbance was used to seed 150 ml nutrient agar to make the assay plates. The agar was swirled to mix thoroughly and poured into large petri dishes which had been placed on a level surface. As soon as the agar was set the plates were placed upside down overnight before using the next day. For assay these seeded plates were removed from 4 C. and allowed to stand at room temperature for 15 min before cutting 7.0 mm diameter wells into the surface of the agar. 250 l of test material (sample or standard) was placed into each well.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] Sample and standard application. All samples and standards were tested in triplicate by adding 250 l to each of 3 wells.

[0287] 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.

[0288] 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).

[0289] Total Activity: all the activity, including activity due to hydrogen peroxide (H.sub.2O.sub.2).

[0290] Non-Peroxide Activity: H.sub.2O.sub.2 is removed by treating samples with catalase enzyme.

[0291] 2. Determination of Honey Activity by H.sub.2O.sub.2 Method

[0292] The activity was measured using the Merckoquant, 1.10011. & 1.10081.

[0293] Peroxide Test Kits. Concentrations expressed as the equivalent mg/L H.sub.2O.sub.2.

[0294] 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.

[0295] 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.

[0296] Results

[0297] 1. Activity Rating

[0298] The antimicrobial activity produced by the modification of the honey samples resulted in a two-fold and almost three-fold respectively increase in phenol activity with PT1 and PT2 compared with Surgihoney alone. The results for the three samples of Surgihoney (SH) and two modified prototypes, PT1 and PT2 are shown in the Table below.

TABLE-US-00008 TABLE 4 The peroxide and non-peroxide antibacterial activities of Surgihoney (SH) and two modified prototypes, PT1 and PT2 against Staphylococcus aureus (NCIMB 9518). Total 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

[0299] 2. Determination of Honey Activity by H.sub.2O.sub.2 Method

[0300] The prototype modifications are observed to generate up to seven and ten times the hydrogen peroxide activity of Surgihoney. The results for the three samples are shown in FIG. 2A. By taking the maximum level of hydrogen peroxide output for each of the three honey prototypes and plotting this against the total phenol activity a linear relationship is observed (FIG. 2B).

[0301] Discussion

[0302] The results from this work show that the main antimicrobial activity of Surgihoney and two modified prototypes, PT1 and PT2 are due to hydrogen peroxide. This is a similar finding to certain other honeys from a variety of floral sources. However, unlike previous work the availability of hydrogen peroxide from the samples is able to be enhanced and at 12 hours is seven and ten times respectively the value for Surgihoney alone. There is a striking linear relationship between the antimicrobial activity and the maximum output of hydrogen peroxide from the three honey prototypes.

[0303] This peroxide activity offers potent antimicrobial activity that is ideally suited to treat or prevent microbial infections. Hydrogen peroxide is an effective antimicrobial and is already used as a biocide for its potent activity against vegetative bacteria, yeasts and spores. It produces its antimicrobial effect through chemical oxidation of cellular components.

[0304] The human toxicity of hydrogen peroxide is concentration dependent and one study has claimed that the differential concentrations for antimicrobial and human toxicity might overlap. By contrast, certain preparations of honey have been shown to be an effective antimicrobial agent by supplying low concentrations of hydrogen peroxide continuously over time rather than as a large amount and without such toxicity. Indeed there is compelling evidence that where physiological levels of hydrogen peroxide are applied to mammalian cells there is a stimulation of biological responses and activation of specific biochemical pathways in these cells.

[0305] Clearly Surgihoney and the two modified prototypes, PT1 and PT2 offer effective hydrogen peroxide release over at least 24 hours.

[0306] Conclusions

[0307] Surgihoney and the two modified prototypes, PT1 and PT2 have been shown to have potent antimicrobial activity against a standard strain of Staphylococcus aureus. These antimicrobial activities have been shown to be due to hydrogen peroxide. The activity is scalable and can be described in terms of hydrogen peroxide activity. These modified honeys are effective, non-toxic and easy to administer.

EXAMPLE 6

[0308] In Vitro Antimicrobial Activity of Surgihoney

[0309] This example describes susceptibility testing of a range of bacterial isolates to Surgihoney by disc diffusion method, minimum inhibitory concentration (MIC) and minimum cidal concentration (MBC) determination, and time bactericidal measurements.

[0310] Summary

[0311] Results: Surgihoney demonstrates highly potent inhibitory and cidal activity against a wide range of Gram positive and Gram negative bacteria and fungi. MIC/MBC's are significantly lower than concentrations likely to be achieved in topical clinical use. Surgihoney 1 MIC/MBC's for Staph. Aureus are 31 and 125 gms/L and Surgihoney 3 MIC/MBC's 0.12 and 0.24 gms/L.

[0312] 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.

[0313] Conclusions: Surgihoney has wide potential as a highly active topical treatment combining the effects of the healing properties of honey with the potent antimicrobial activity of the bioengineered product. It is highly active against multidrug resistant bacteria. It is more active than other honeys tested and comparable to chemical antiseptics in antimicrobial activity.

[0314] 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.

[0315] Methods

[0316] 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.

[0317] Clinical isolates were collected from soft tissue microbiology samples. Eighteen isolates of Staphylococcus aureus, 12 methicillin-sensitive (MSSA) and 6 methicillin-resistant (MRSA), 6 isolates of haemolytic streptococci, Lancefield groups A (2), B (2), C (1), G (1), 5 isolates of Enterococcus spp. Including vancomycin-resistant E. faecium, 6 of Esch. coli, including extended spectrum lactamase producers, 2 of Klebsiella spp., 1 Serratia Marcescens Amp C producer, 4 of Pseudomonas aeruginosa, 1 of Acinetobacter 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.

[0318] Agar Diffusion

[0319] 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.

[0320] 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 (Iodoflex). Wells were cut in the plates inoculated with Staphylococcus aureus and filled with test honey or in the case of the dressings, these were cut to 22 cm and placed on the surface of the inoculated plates.

[0321] 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.)

[0322] Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations

[0323] Surgihoney product was warmed to 37 C. to liquefy it and 5 gms was mixed with 10 mL sterile deionised water. This dilution was regarded as the neat substance for serial dilution. The British Society of Antimicrobial Chemotherapy (BSAC) method for performing minimum inhibitory concentrations (MIC's) and minimum bactericidal concentrations (MBC's) was used (Andrews J M. Determination of minimum inhibitory concentrations. J Antimicrob 372 Chemother 2001; 48(Supp 1): 5-16). The Surgihoney products were serially diluted in microtitre tray wells from neat to 1 in 1024. 75 L of each honey dilution was added to each well in the strip of the microtitre tray. The neat concentration represented a concentration of 250 gm/L and the 1 in 2048 dilution, approximately 0.12 gm/L.

[0324] 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.

[0325] All wells including controls were inoculated with 75 L of the test isolate preparation. The well trays were incubated at 37 C. for 18 hours. The MIC was regarded as the most dilute well that showed no detectable turbidity.

[0326] 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.

[0327] Time Kill Curves

[0328] 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.

[0329] 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.

[0330] 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.

[0331] Results

[0332] Inhibitory Zone Sizes.

[0333] 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).

[0334] In the quantitative zone size testing, Surgihoney at all potencies produced an inhibitory zone in agar diffusion against all bacteria tested, both Gram positive and Gram negative bacteria including multiply antibiotic resistant bacteria, and fungal species. The zone size for each species increased with increasing Surgihoney potency preparations. Table 5. The inhibitory effect of Surgihoney was not dependant only on direct contact with the active agent as with the silver dressings, but diffused well beyond the well producing the extensive zones listed in Table 5.

[0335] MIC's & MBC's

[0336] Surgihoney demonstrated significant antimicrobial activity against all the isolates tested. MIC's and MBC's were very consistent amongst isolates of the same species whether the isolates were multidrug resistant or highly sensitive. Table 6 lists the MIC and MBC values for isolate species tested by dilution ratio and Table 7 shows the MIC and MBC's in grams per litre. The degree of potency rose with the grade of Surgihoney. The MBC for each isolate was close to the MIC within a single dilution in most cases.

[0337] Topical concentration of Surgihoney is estimated at approximately 500 gms/L. Surgihoney 1 MIC/MBC's for Staph. Aureus are 31 and 125 gms/L and Surgihoney 3 MIC/MBC's 0.12 and 0.24 gms/L respectively.

[0338] Time Kill Curves.

[0339] Surgihoney kills bacteria rapidly. Starting with a colony forming units per millilitre (cfu/mL) of approximately 105, cfu/mL numbers in the control rose steadily, whereas in the Surgihoney inocula the cfu/mL fell rapidly after contact with both potencies of Surgihoney. By 30 minutes cfu numbers had fallen 1000 fold in most cases for both S1 and S3 (FIG. 3). For S1 bacterial growth was undetectable by 2 hours in most cases and for S3 by 30 minutes. Enterococci appeared more resilient and persisted for 48 hours. Cidal activity was complete for all organisms as terminal culture in nutrient broth with subsequent plating on blood agar failed to detect any organism in the S1 or S3 inocula.

[0340] Discussion

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] These in vitro studies have demonstrated the potential of Surgihoney with high antimicrobial activity whose potency can be controlled.

[0346] Conclusion

[0347] These in vitro results support the clinical use of Surgihoney as a potent and non-toxic antimicrobial.

TABLE-US-00009 TABLE 5 Inhibitory zones sizes with different potencies of Surgihoney (S1, S2, S3) S1 S2 S3 No. of Mean zone Mean zone 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 1 32.1 43.7 55.2 lwoffii Bacteroides fragilis 1 22.3 28.7 34.2 Propionibacterium 1 19.7 23.4 31.9 acnes Candida sp. 2 9 (8-10) 15 (15) 26 (24-28) Aspergillus 1 8 12 18 fumigatus

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

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

EXAMPLE 7

[0348] Surgihoney Emulsion

[0349] Preparation

[0350] 10 g Surgihoney was dissolved in 10 ml of glycerol. 10 ml of paraffin oil was then added to a Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 1 ml of PGPR (Polyglycerol polyricinoleate) was then added. The rheometer was then started under the following conditions; Shear rate 2000 1/s, Temperature set at 37.5 C. After 2 minutes, 10 ml of Surgihoney-glycerol solution was added dropwise. Once a total of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier to a container.

[0351] Optical Microscopy

[0352] Optical microscopy revealed that the emulsion contained reverse micelles which encapsulated Surgihoney. Such micelles can be observed in FIG. 4. The average micelle diameter was found to be 178 m.

[0353] Hydrogen Peroxide Tests

[0354] Hydrogen peroxide stick tests (Purchased from Sigma Aldrich (Quantofix)) were used to detect hydrogen peroxide in the emulsion. The tests were carried out before and after addition of water, and showed that before addition of water, the emulsion produced no hydrogen peroxide, and after water was added, the emulsion tested positive for hydrogen peroxide. A positive test was indicated by a colour change to blue.

[0355] Stability Test

[0356] The emulsion maintained its capacity to generate hydrogen peroxide after storage at ambient conditions for at least four weeks.

[0357] Spray Test

[0358] The emulsion was added to a pump-action spray bottle and was found to be sprayable.

EXAMPLE 8

[0359] Effects of Different Parameters on Stability of Surgihoney Emulsions

[0360] The effects of changing the Surgihoney emulsion preparation method described in Example 7, one parameter at a time, were investigated. The changes and their effects are summarised below.

[0361] i) Proportion of the Oil Phase to the Surgihoney-Glycerol Phase

[0362] Oil volumes greater than 10 ml, and less than 10 ml, were tested. The emulsion was found to be more stable when a lower volume of oil was used compared to the volume of the Surgihoney-glycerol phase. When the volume of the oil was less than 6 ml, the emulsion was found to separate by less than 3% in total volume over 72 hours. A volume of 4 ml allowed a separation of just 1.3% of the total volume over 72 hours. This stability is far greater than that of the method described in Example 7, which provided an emulsion with a separation of 9.4% of total volume over the same time period.

[0363] ii) Volume of PGPR

[0364] PGPR volumes up to 4 ml, and less than 1 ml, were tested. The emulsion was more stable when a higher amount of PGPR was used. At a volume of 4 ml, PGPR provided greater stability than with use of lower volumes, and far greater stability than that of the emulsion described in Example 7.

[0365] iii) Shear Rate

[0366] Shear rates from 1000 1/s to 3000 1/s were tested. The emulsion was more stable when a higher shear rate was applied. A shear rate of 3000 1/s produced the most stable emulsion. Separation of only 4.6% of the total volume over 72 hours was observed for emulsion prepared at this shear rate, compared with a separation volume of 9.4% of the total volume over the same time period for emulsion prepared as described in Example 7.

[0367] iv) Temperature

[0368] Temperatures from 20 C. to 40 C. were tested. There was no noticeable trend regard the stability of the emulsions as temperature was increased. However a temperature of 40 C. produced the most stable emulsion. Separation of only 3.1% of the total volume over 72 hours was observed for this emulsion.

[0369] v) Length of Shear

[0370] Shear times of 20 minutes and 30 minutes were tested, in addition to that used in the preparation method described in Example 7. However, there was no significant difference produced by extending the shear time.

[0371] vi) Order of Reagent Addition

[0372] The effect of changing the order in which the reagents are added to the rheometer was tested. The effect of adding all of the components before starting the rheometer was compared with the effect of adding the Surgihoney-glycerol and oil components first, then adding the PGPR after 1-2 minutes. The most stable emulsion was formed when the PGPR was added last. The resulting emulsion provided a separation volume of 2.8% of the total volume over 120 hours.

[0373] vii) Concentration of Surgihoney Dissolved in Glycerol

[0374] The following ratios of Surgihoney (g) to glycerol (ml) were tested: 1 g:1 ml; 0.5 g:1 ml; 2 g:1 ml. The ratio that produced the most stable emulsion was 1 g:1 ml, the same ratio used in the preparation method described in Example 7.

[0375] viii) Sodium Chloride

[0376] When sodium chloride is dissolved in the polar layer of the emulsion, it increases the polarity of this layer. It also forms electrostatic interactions with the lipid layer of the emulsion. The electrostatic interactions and increased polarity could improve stability and reduce coalescence. However, addition of sodium chloride (1 g, 2 g or 4 g) was not found to influence the stability of the emulsion.

[0377] The effects of the changes are summarised in the table below:

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

EXAMPLE 9

[0378] Surgihoney Emulsions with High Stability

[0379] The results from the changes described in Example 8 were used to design a further method of preparing Surgihoney emulsion. This method is described below.

[0380] Preparation 10 g Surgihoney was dissolved in 10 ml of glycerol. 4, 6, 8, or 10 ml of Paraffin oil was then added to the rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 10 ml of Surgihoney-glycerol solution was then added to the rheometer. The rheometer was then started under the following conditions; Shear rate 3000 1/s, Temperature 40 C., gap 4000 m, Run time 10 minutes. After 1 minute 4 ml of PGPR (Polyglycerol polyricinoleate) was then added. Once a total of 10 minutes had elapsed the emulsion was transferred from the rheometer to a container.

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

[0381] All of the formulations were found to be highly stable, with a slight increase in stability observed as the volume of paraffin oil used was increased.

EXAMPLE 10

[0382] Surgihoney Cream Formulation

[0383] 1.5 g of Surgihoney was dissolved in 1.5 ml of glycerol. 1 g of sodium alginate was then dissolved in the Surgihoney-glycerol solution. Next 10 ml of Paraffin oil was added to the Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 1 ml of PGPR (Polyglycerol polyricinoleate) was then added. The rheometer was then started under the following conditions; Shear rate 2000 1/s, Temperature set at 37.5 C., gap 4000 m, Run time 10 minutes. After 2 minutes, 1.5 ml of the Surgihoney-alginate and glycerol solution was added to the rheometer. After 3 minutes 8 ml of calcium chloride solution was added dropwise to the rheometer. Once a total of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier to a container.

EXAMPLE 11

[0384] Non-Aqueous Surgihoney Cream Formulation

[0385] The method described in Example 10 uses water to dissociate calcium chloride into its ions. This could potentially activate the Surgihoney to produce hydrogen peroxide, and limit the stability of the cream formulation. However, we have appreciated that calcium chloride can be dissociated using non-aqueous solvents, such as ethanol or acetic acid. We have also appreciated that glycerol is able to bind to free water. This property allows water to be used to dissolve the alginate, provided sufficient glycerol is present to prevent premature release of hydrogen peroxide.

[0386] The method described below uses ethanol as a solvent for calcium chloride, and glycerol to bind free water in the alginate solution.

[0387] 1 g of sodium alginate is dissolved in 15 ml water. Next 30 ml glycerol is added to the alginate solution and mixed. Then 30 g Surgihoney is then dissolved in the solution. 10 ml of Paraffin oil is then added to the rheometer (TA Instruments AR-G2) which has a Jacket Peltier and vane geometry attached. 10 ml of Surgihoney solution is then added to the rheometer. The rheometer is then started under the following conditions: Shear rate 3000 1/s, Temperature 40 C., gap 4000 m, Run time 10 minutes. After 1 minute 4 ml of PGPR (Polyglycerol polyricinoleate) is added. After 2 minutes 8 ml of non-aqueous calcium chloride solution (1M Calcium chloride in ethanol) is added dropwise to the rheometer. Once a total of 10 minutes has elapsed, the emulsion is transferred from the rheometer to a container.

[0388] Summary of emulsion formulations in Examples 7-11:

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