Antimicrobial superabsorbent compositions

Abstract

A composition has an enzyme that is able to convert a substrate to release hydrogen peroxide; a substrate for the enzyme; and a superabsorbent component, such as a superabsorbent polymer. The composition is in the form of a powder and may form a gel on contact with water.

Claims

1. A sterile composition comprising: an enzyme that is able to convert a substrate to release hydrogen peroxide; a substrate for the enzyme; and a superabsorbent component, wherein the composition is in the form of a powder, wherein the powder has a mean and/or modal particle diameter of 3000 μm or less, and wherein the composition does not contain any peroxidase.

2. A composition according to claim 1, wherein the superabsorbent component has an absorption capacity of at least 10 g/g.

3. A composition according to claim 1, wherein the absorption capacity is in respect of deionized water, distilled water, or a 0.9% (by weight) saline solution.

4. A composition according to claim 1, wherein the superabsorbent component is a superabsorbent polymer.

5. A composition according to claim 4, wherein: a) the superabsorbent polymer is anionic; and/or b) the superabsorbent polymer is cross-linked; and/or c) the superabsorbent polymer is polyacrylate or polyacrylamide.

6. A composition according to claim 4, wherein the superabsorbent polymer is selected from the group consisting of: a hydrolysed cellulose-polyacrylonitrile; a starch-polyacrylonitrile co-polymer; a cross-linked co-polymer of maleic anhydride; polyvinyl alcohol co-polymer; and cross-linked polyethylene oxide.

7. A composition according to claim 1, wherein the powder has a mean and/or modal particle diameter of 100 μm to 2000 μm.

8. A composition according to claim 1, wherein the enzyme is an oxidoreductase enzyme.

9. A composition according to claim 1, wherein the substrate is a sugar.

10. A composition according to claim 1, comprising a solute in the form of a sugar or sugar derivative having a solubility of at least 100 g/100 g water at 20° C. and 1 atm.

11. A composition according to claim 1, wherein the composition: a) Lacks catalase activity; and/or b) Comprises a blood clotting agent; and/or c) Does not contain zinc oxide; and/or d) Comprises a coagulation factor.

12. A composition according to claim 1, comprising a freeze-drying protective agent.

13. A wound dressing which comprises a dressing material and a composition according to claim 1.

14. An airtight container comprising a composition according to claim 1, the container comprising a one or a plurality of apertures for permitting dispensing of the composition, the one or plurality of apertures being sealed or covered prior to use.

15. A method for making a composition according to claim 1, comprising contacting a superabsorbent component with an enzyme that is able to convert a substrate to release hydrogen peroxide and a substrate for the enzyme.

16. A method of preventing, treating, or ameliorating a microbial infection, which comprises administering a composition according to claim 1, to a subject in need of such prevention, treatment or amelioration.

17. A method of treating a wound, which comprises administering a composition according to claim 1 to a wound site.

Description

(1) Preferred embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

(2) FIG. 1 shows a photograph of a superabsorbent powder according to an embodiment of the invention, prior to addition of water;

(3) FIG. 2 shows the resulting gel following addition of 15 ml of water to a superabsorbent powder according to an embodiment of the invention, with a hydrogen peroxide test strip indicating hydrogen peroxide production;

(4) FIG. 3 shows the gel of FIG. 2, in an inverted orientation.

(5) FIG. 4 shows a gel following addition of 30 ml of water to a superabsorbent powder according to an embodiment of the invention;

(6) FIG. 5 shows the gel of FIG. 4 in an inverted orientation;

(7) FIG. 6 shows the particle size distributions of examples of superabsorbent powder compositions of the invention;

(8) FIG. 7 shows a % strain sweep depicting the LVR for both extremes of current formulations including formulations which include maltodextrin and methylated β-Cyclodextrin;

(9) FIG. 8 the effect of dilution upon the physical properties of examples of gels of the invention;

(10) FIG. 9 shows the effect of increasing the amount of cross-linked sodium polyacrylate on viscoelastic properties of examples of gels of the invention;

(11) FIG. 10 shows the effect of dilution at a fixed frequency (1 Hz) upon the storage modulus of examples of gels of the invention;

(12) FIG. 11 shows a comparison of storage modulus values at different dilutions between different examples of gels of the invention;

(13) FIG. 12 is a graph showing the effect of compositions suitable for use in forming examples of superabsorbent powders of the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to Surgihoney™, at various concentrations;

(14) FIG. 13 is a graph showing the effect of sterile and non-sterile compositions suitable for use in forming examples of superabsorbent powders of the invention, comprising glucose, glucose oxidase and fructose (SyntheticRO) (buffered at pH 4.03) on the growth of planktonic MRSA, at various concentrations;

(15) FIG. 14 is a graph showing the effect of sterile and non-sterile compositions suitable for use in forming examples of superabsorbent powders of the invention, comprising glucose, glucose oxidase and fructose (SyntheticRO) (unbuffered) on the growth of planktonic MRSA, at various concentrations;

(16) FIG. 15 is a graph showing the effect of sterile and non-sterile compositions suitable for use in forming examples of superabsorbent powders of the invention, comprising glucose, glucose oxidase and fructose (SyntheticRO) (buffered at pH 7.04) on the growth of planktonic MRSA, at various concentrations;

(17) FIG. 16 is a table showing the effect of sterile and non-sterile compositions suitable for use in forming examples of superabsorbent powders of the invention comprising glucose, glucose oxidase and fructose, on the MIC and MBC of planktonic MRSA, at various concentrations;

(18) FIG. 17 shows the effect of compositions suitable for use in forming examples of superabsorbent powders of the invention, comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

(19) FIG. 18 shows the effect of SyntheticRO on the MIC and MBC of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

SPECIFIC EXAMPLES

Example 1—Synthesis of Superabsorbent Powder

(20) SurgihoneyRO (Sachet) or SyntheticRO (a mixture of purified glucose oxidase, purified glucose and purified fructose; see example 5) and either methylated β-Cyclodextrin (CycloLab R&D, UK) or Maltodextrin (Sigma Aldrich, UK) were first weighed out in equal proportions (50:50 ratio).

(21) The desired amount (0.5, 1, 2 or 3 g) of cross-linked sodium polyacrylate (Sigma Aldrich, UK) was also weighed out.

(22) All three components were then mixed together until a homogenous formulation was achieved. This formulation was then placed inside a freezer mill tube and submerged in liquid nitrogen for approximately 1 minute. The freezer mill tube was then placed in the freezer mill (SPEX SamplePrep). The freezer mill was run at a rate of 30 cycles per second for a total time of 3 minutes.

(23) For compositions containing SurgihoneyRO, it was found that milling the mixture first allowed for a more complete drying process.

(24) The samples were then transferred into the freeze drying chamber (Frozen in Time). The freeze drier cold trap was set to −55° C. and was at a pressure of 4.0×10.sup.−4 mbar. The sample was freeze dried for a total of 48 hours.

(25) The samples were removed and sieved using 1000 μm and 500 μm gratings. Any powder that remained in the sieves was crushed using a pestle and mortar and ran through the sieves again. The powder was then sealed in a pot containing silica gel beads which act as a desiccant.

Example 2—Formation of Gels

(26) To demonstrate formation of gels, water was added to a powder containing 40:60 SurgihoneyRO™ to cyclodextrin composition containing 3 wt % sodium polyacrylate.

(27) FIG. 1 shows 1 g of the powder prior to addition of water, in a weighing boat.

(28) FIG. 2 shows the resulting gel following addition of 15 ml of water. It also shows a hydrogen peroxide test strip indicating the production of hydrogen peroxide by the gel. FIG. 3 shows the same gel, but in an inverted orientation. This demonstrates how the gel may readily adhere to a surface.

(29) FIG. 4 shows the gel following addition of 30 ml of water, and FIG. 5 shows the same gel in an inverted orientation.

Example 3—Particle Size Analysis

(30) An instrument called QICPIC (Sympatec, UK) was employed, which uses of dynamic image analysis to size particles. High speed image analysis used a pulsed light source with illumination times of less than 1 nanosecond. The particles are optically frozen while a high-resolution, high-speed camera captures the particle projections. Algorithms built into the instrument software evaluate the particles giving statistically relevant results. The following parameters were used in order to determine particle size: Calculation mode: EQPC—this mode is used to calculate the diameter of a circle that has the same area as the projection area of the particle. Trigger condition: start 0s, valid 300 s Dispersing method: 2 mm 25% 4 mm Frame rate: 100 fps Height of fall: 40.00 cm Feeder: Vibrate Feed rate: 25.00% Gap height: 2.00 mm

(31) The amount of superabsorbent polymer in the compositions and the type of freeze-drying protective agent was varied in order to establish the impact of varying these parameters on the properties of the compositions.

(32) The following compositions were tested, and their peak particle distributions and sphericities were established. A superabsorbent reactive oxygen powder containing 47.6 wt. % methylated β-Cyclodextrin, 47.6 wt. % SurgihoneyRO and 4.8 wt. % cross-linked sodium polyacrylate. This composition contains a peak particle size distribution at 204.5 μm and a peak sphericity distribution at 0.9. A superabsorbent reactive oxygen powder containing 45.5 wt. % methylated β-Cyclodextrin, 45.5 wt. % SurgihoneyRO and 9.1 wt. % cross-linked sodium polyacrylate. This composition contains a peak particle size distribution at 204.5 μm and a peak sphericity distribution at 0.9. A superabsorbent reactive oxygen powder containing 41.7 wt. % methylated β-Cyclodextrin, 41.7 wt. % SurgihoneyRO and 16.7 wt. % cross-linked sodium polyacrylate. This composition contains a peak particle size distribution at 186.4 μm and a peak sphericity distribution at 0.9. A superabsorbent reactive oxygen powder containing 38.5 wt. % methylated β-Cyclodextrin, 38.5 wt. % SurgihoneyRO and 23.1 wt. % cross-linked sodium polyacrylate. This composition contains a peak particle size distribution at 204.5 μm and a peak sphericity distribution at 0.9. A superabsorbent reactive oxygen powder containing 38.5% methylated β-Cyclodextrin, 38.5% wt. % SyntheticRO and 23.1 wt. % cross-linked sodium polyacrylate This composition contains a peak particle size distribution at 540.5 μm and a peak sphericity distribution at 0.82. A superabsorbent reactive oxygen powder containing 47.6 wt. % maltodextrin, 47.6 wt. % SurgihoneyRO and 4.8 wt. % cross-linked sodium polyacrylate. This composition contains a peak particle size distribution at 405.7 μm and a peak sphericity distribution at 0.85. A superabsorbent reactive oxygen powder containing 45.5 wt. % maltodextrin, 45.5 wt. % SurgihoneyRO and 9.1 wt. % cross-linked sodium polyacrylate. This composition contains a binomial distribution with peak particle size distributions at 445.0 μm and 2107.4 μm, a peak sphericity distribution at 0.85. A superabsorbent reactive oxygen powder containing 41.7 wt. % maltodextrin, 41.7 wt. % SurgihoneyRO and 16.7 wt. % cross-linked sodium polyacrylate, This composition contains a peak particle size distribution at 445.0 μm a peak sphericity distribution at 0.85. A superabsorbent reactive oxygen powder containing 38.5 wt,% maltodextrin, 38.5 wt. % SurgihoneyRO and 23.1 wt. % cross-linked sodium polyacrylate. This composition contains a binomial distribution with peak particle size distributions at 445.0 μm and 2107.4 μm a peak sphericity distribution at 0.85. A superabsorbent reactive oxygen powder containing 38.5 wt. % maltodextrin, 38.5 wt. % SyntheticRO and 23.1 wt. % cross-linked sodium polyacrylate. The composition contains a polymodal distribution with peak particle size distributions at 540.5 μm, 968.4 μm and 1428.5 μm; and two peak sphericity distribution at 0.45 and 0.85.

(33) FIG. 6 a) illustrates the particle size distributions of each composition containing β-cyclodextrin sodium polyacrylate and SurgihoneyRO/SyntheticRO. FIG. 6 b) illustrates the particle size distributions of each composition containing maltodextrin, sodium polyacrylate and SurgihoneyRO/SyntheticRO.

(34) The results obtained suggest the following: Increasing the wt % of cross-linked sodium polyacrylate may not affect particle size. Compositions containing methylated β-cyclodextrin may have a smaller average particle size than those that contain maltodextrin. Formulations that contain methylated β-cyclodextrin may produce more spherical particles. Formulations containing Maltodextrin appear to aggregate or agglomerate to form larger particles post-sieving.

Example 4—Gel Rheology Analysis

(35) Firstly the linear viscoelastic region (LVR) was determined for all of the tested powder gels. The LVR is determined so that the material behaves linearly and an out of phase sinusoidal shear stress response is produced. This was achieved by conducting a strain sweep on a Rheometer (TA Instruments, UK) using a 40 mm steel sand blasted plate geometry in parallel to another sand blasted steel plate.

(36) FIG. 7 shows a % strain sweep depicting the LVR for both extremes of tested formulations including formulations which include maltodextrin (MD) and methylated β-Cyclodextrin (CD).

(37) The strain sweep was conducted under the following conditions: % strain range was set logarithmically from 0.01 to 50.0 with 10 points measured per decade. Temperature set to 34° C. relevant to the final topical application with an equilibration time of 2 minutes. The frequency was kept constant at 1 Hz Geometry gap was set at 1000 μm

(38) The strain sweep identified a % strain that fell into the LVR for all of the samples; this was found to be 0.5% strain.

(39) Frequency sweeps were then carried out at this % strain and the following conditions: Frequency range was set logarithmically from 0.1 and 100 Hz with 10 points measured per decade. Temperature set to 34° C. relevant to the final topical application with an equilibration time of 2 minutes.

(40) The same compositions as those in Example 3 were used to form gels by adding varying amounts of water (5 ml, 10 ml or 20 ml).

(41) FIG. 8 a) shows the effect of dilution upon the physical properties of the gel formed from a composition containing 38.5 wt. % methylated β-cyclodextrin, 38.5 wt. % SurgihoneyRO and 23.1 wt. % cross-linked sodium polyacrylate, at a range of frequencies. FIG. 8 b) shows the same composition but with syntheticRO, rather than SurgihoneyRO.

(42) The results indicate how shows how the gels formed may be more elastic than viscous (higher G′ (storage modulus) than G″ (loss modulus)), over the range of frequencies; and how there may be a low frequency dependency. Similar effects were observed for the other compositions.

(43) FIG. 9 shows the effect of increasing the amount of cross-linked sodium polyacrylate in maltodextrin: SurgihoneyRO/SyntheticRO compositions containing 4.8 wt. %, 9.1 wt. %, 16.7 wt. % and 23.1 wt. % upon the storage modulus (G′). The gels were formed by adding 5 ml of distilled water. FIG. 9 shows how an increase in cross-linked sodium polyacrylate may increase the stiffness of the gel that is formed. Similar effects were observed for the other compositions.

(44) FIG. 10 shows the effect of dilution at a fixed frequency (1 Hz) upon the storage modulus of the gel containing varying amounts of cross-linked sodium polyacrylate in maltodextrin: SurgihoneyRO/SyntheticRO formulations.

(45) FIG. 11 a) shows a comparison of storage modulus values at different dilutions between gels formed from compositions containing maltodextrin: SurgihoneyRO and compositions containing methylated β-cyclodextrin: SurgihoneyRO, the compositions containing 16.7 wt. % cross-linked sodium polyacrylate. FIG. 11 b) shows results from the same formulations, but which contain SyntheticRO rather than SurgihoneyRO.

(46) The results obtained suggest the following: An increase in cross-linked sodium polyacrylate may increase the stiffness of the gel; There is a low frequency dependency; Samples may be more elastic than viscous; Gels containing SyntheticRO may have a higher G′ and G″ than those which contain SurgihoneyRO; There may be no significant difference between the rheology of the formulations that contain maltodextrin or cyclodextrin; Cyclodextrin-based formulations may dissolve more readily upon application to water than those which contain maltodextrin.

Example 5—Synthetic Honey Compositions (Also Known as SyntheticRO)

(47) Samples with batch number “RO” contain no glucose oxidase.

(48) Samples with batch number “RO1” contain 50 ppm glucose oxidase.

(49) Samples with batch number “RO2” contain 1000 ppm glucose oxidase.

(50) A. pH 4.03 Buffered Samples

(51) A1. Batch no NB01p43RO Non sterile

(52) TABLE-US-00001 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

(53) Description

(54) Non sterile base buffered saccharide solution.

(55) A2. Batch no NB01p43RO Sterile

(56) TABLE-US-00002 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

(57) Description Sterile base buffered saccharide solution

(58) A3. Batch no NB01p44RO1 Non sterile

(59) TABLE-US-00003 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

(60) Description

(61) Non sterile base buffered RO1 saccharide solution.

(62) A4. Batch no NB01p44RO1 Sterile

(63) TABLE-US-00004 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

(64) Description

(65) Sterile base buffered RO1 saccharide solution

(66) A5. Batch no NB01p44RO2 Non sterile

(67) TABLE-US-00005 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

(68) Description

(69) Non sterile base buffered RO2 saccharide solution.

(70) A6. Batch no NB01p43RO2 Sterile

(71) TABLE-US-00006 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0% GOX enzyme N/A

(72) Description Sterile base buffered RO2 saccharide solution

(73) B. Unbuffered Samples

(74) B1. Batch no NB01p51RO Non sterile

(75) TABLE-US-00007 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

(76) Description

(77) Non sterile base buffered saccharide solution.

(78) B2. Batch no NB01p51RO Sterile

(79) TABLE-US-00008 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

(80) Description Sterile base buffered saccharide solution

(81) B3. Batch no NB01p51RO1 Non sterile

(82) TABLE-US-00009 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

(83) Description

(84) Non sterile base buffered RO1 saccharide solution.

(85) B4. Batch no NB01p51RO1 Sterile

(86) TABLE-US-00010 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

(87) Description

(88) Sterile base buffered RO1 saccharide solution

(89) B5. Batch no NB01p51RO2 Non sterile

(90) TABLE-US-00011 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17 0%

(91) Description

(92) Non sterile base buffered RO2 saccharide solution. B6. Batch no NB01p51RO2

(93) Sterile

(94) TABLE-US-00012 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

(95) Description

(96) Sterile base buffered RO2 saccharide solution

(97) C. pH 7.04 Buffered Samples

(98) C1. Batch no NB01p57RO Non sterile

(99) TABLE-US-00013 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

(100) Description

(101) Non sterile base buffered saccharide solution.

(102) C2. Batch no NB01p57RO Sterile

(103) TABLE-US-00014 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

(104) Description

(105) Sterile base buffered saccharide solution

(106) C3. Batch no NB01p57RO1 Non sterile

(107) TABLE-US-00015 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

(108) Description

(109) Non sterile base buffered RO1 saccharide solution.

(110) C4. Batch no NB01p57RO1 Sterile

(111) TABLE-US-00016 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

(112) Description

(113) Sterile base buffered RO1 saccharide solution

(114) C5. Batch no NB01p57RO2 Non sterile

(115) TABLE-US-00017 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

(116) Description

(117) Non sterile base buffered RO2 saccharide solution.

(118) C6. Batch no NB01p57RO2

(119) TABLE-US-00018 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

(120) Description

(121) Sterile base buffered RO2 saccharide solution

Example 6—Efficacy of Synthetic Honey Compositions Against Planktonic MRSA

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

(123) The results are shown in FIGS. 1 to 5.

(124) The results show that, like Surgihoney, synthetic compositions containing glucose, glucose oxidase and fructose are able to inhibit microbial growth.

(125) Out of all of synthetic compositions, the synthetic composition buffered at pH7.04 had the most effective MIC. Sterilised compositions were more effective than non-sterilised compositions, and synthetic composition buffered at pH7.04 synthetic had the most effective MBC when compared to other synthetic compositions and even when compared to SurgihoneyRO.

(126) FIGS. 17 (a to d) and 18 show MIC and MBC results including SurgihoneyRO (RO2) samples and synthetic RO2 samples.