Antimicrobial Composition

Abstract

A composition comprising a hydrogen peroxide source and at least one metal halide. The hydrogen peroxide source comprises hydrogen peroxide and a means for generating hydrogen peroxide. The means for producing hydrogen peroxide comprises at least one oxidoreductase and at least one oxidoreductase substrate. The oxidoreductase substrate comprises at least one sugar, said sugar located within the composition. The composition is held under conditions that render the components inactive until rehydrated.

Claims

1. A composition comprising: a hydrogen peroxide source; and at least one metal halide; wherein the hydrogen peroxide source comprises hydrogen peroxide; and a means for producing hydrogen peroxide; wherein the means for producing hydrogen peroxide comprises at least one oxidoreductase; and at least one oxidoreductase substrate wherein the oxidoreductase substrate comprises at least one sugar, said sugar located within the composition; and wherein the composition is held under conditions that render the components inactive until rehydrated.

2. The composition according to claim 1 wherein the metal halide is selected from a metal chloride, metal fluoride, metal iodide, metal bromide and any mixtures thereof.

3. The composition according to claim 1 wherein the metal chloride is selected from magnesium chloride, calcium chloride, potassium chloride, sodium chloride, lithium chloride, nickel chloride, silver chloride, ferric/ferrous chloride, potassium chloride, hydrogen chloride, copper chloride, chromium chloride manganese chloride, cobalt chloride, zinc chloride, barium chloride, beryllium chloride, cadmium chloride, aluminium chloride, gold chloride, titanium chloride and any ions, salts, isomers or any mixtures thereof.

4. The composition according to claim 3 wherein the metal chloride is sodium chloride.

5. The composition according to claim 1 further includes 8-methoxy psoralen.

6. The composition according to claim 1, wherein the oxidoreductase substrate is D-Glucose.

7. The composition according to claim 1, wherein the hydrogen peroxide source is in solution.

8. The composition according to claim 7, wherein the solution is aqueous.

9. The composition according to claim 1, wherein the composition comprises a solvent.

10. The composition according to claim 9, wherein the solvent is water.

11. The composition according to claim 1, wherein the composition has a pH of from approximately 3 to 8.

12. The composition according to claim 1 wherein the hydrogen peroxide source comprises glucose oxidase, D-glucose, additional sugars selected from one or more of sucrose, fructose and/or maltose, and hydrogen peroxide in an aqueous/non aqueous solution; wherein glucose oxidase is present at an activity of at least 10 U per 100 g of the composition; D-glucose is present from 20 to 85% by weight based on the weight of the total composition; additional sugars selected from one or more of sucrose, fructose and/or maltose are present from 5 to 70% by weight based on the weight of the total composition; water or another solvent is present from 10 to 20% by weight based on the weight of the total composition; the composition has a pH from approximately 3 to 8; and wherein the composition provides a two-stage hydrogen peroxide release in which (a) hydrogen peroxide is available for immediate release from the composition at a level of at least 0.1 mg per litre; and (b) the sustained release of further hydrogen peroxide for at least a twenty-four hour period occurs upon rehydration of the composition.

13. A medicament comprising a composition according to claim 1.

14. The medicament according to claim 13, wherein the medicament comprises a topical delivery system suitable for topical administration of the composition according to claim 1.

15. The medicament according to claim 13 wherein the medicament comprises an enteral delivery system suitable for oral administration of the composition according to claim 1.

16. The medicament according to claim 13, wherein the medicament comprising a parenteral delivery system suitable for injection administration of the composition according to claim 1.

17. The composition according to claim 1 for use as a medicament.

18. The composition according to claim 17 for use as an antimicrobial.

19. The composition according to claim 17 for use in the treatment or prophylaxis of fungal nail.

20. The composition according to claim 17 for use in the treatment or prophylaxis of a Campylobacter infection.

21. The composition according to claim 17 for use in the treatment or prophylaxis of Cryptosporidium infections.

22. The composition according to claim 1 for use as a medicament in the treatment of wounds, infectious keratitis, collagen deficiency disorders, colony collapse disorder/pesticide detoxification in bees, methane reduction in ruminants, bacterial vaginosis, biofilm removal, mastitis, induction of hermetic effects and use as a preservative for foodstuffs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The invention will be more clearly understood by the following description of some embodiments thereof, given by way of example only with reference to the accompanying drawings, in which:

[0078] FIG. 1 is graph showing catalase inhibition by sodium chloride;

[0079] FIG. 2 is a graph showing the effects of metal halides on GOX activity;

[0080] FIG. 3 is a bar chart showing percentage (%) performance improvement against various species of micro-organisms when A.sup.3IS is combined with sodium chloride; and,

[0081] FIG. 4 is a bar chart showing the effectiveness of A.sup.3IS and A.sup.3IS+sodium chloride against the most drug resistant pathogens as identified by WHO.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] Referring to FIG. 1 the known effect of sodium chloride on catalase activity, changes in leaf catalase activity, from P. vulgaris (.square-solid.) and M. sativa (□), during in vitro incubation (1 h) in different NaCl doses is shown. Values, means of four independent assays, ±SE, are expressed as percentage relative to the activity without NaCl (P. vulgaris, 0.36 μkat mL.sup.−1; M. sativa, 1.35 μkat mL.sup.−1).

[0083] Referring now to FIG. 2 the known effects of metal chlorides on the GOX activity at pH 5.6. Sodium chloride, +; potassium chloride, ◯; calcium chloride, ∨; and magnesium chloride is shown.

The test formulations begin at 500 mM to 1000 mM where there is no evidence of GOX activation. The effect of halide ions on the activity of the glucose oxidase enzyme indicates that a significant reduction in the activity should be expected.

[0084] Referring now to FIG. 3 the combined effect of sodium chloride (NaCl) with A.sup.3IS against various species of microorganism is shown. Sodium chloride was added to the combination at 0.5M or 1M. The addition of NaCl saw a significant improvement of performance across all species of microorganisms tested against A.sup.3IS alone (control; 0M NaCl).

[0085] Referring now to FIG. 4 the effect of A.sup.3IS alone and in combination with sodium chloride against various drug resistant pathogens is shown.

EXAMPLES

Example 1: General Materials and Methods

[0086] Kirby-Bauer Antibiotic Testing

[0087] The purpose of the Kirby-Bauer disk diffusion susceptibility test is to determine the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds.

TABLE-US-00002 TABLE 1 Drug Class presence Antibiotic WHO priority of catalase Concentration (ug/disk) pathogen + E. coli Critical High Medium + K. pneumonia + Acinetobacter beijerinckii + Acinetobacter baumannii + P. aeruginosa + S. aureus (MR) − E. faecalis + C. jejuni + Neisseria gonorrhoeae MR = methicilin resistant

[0088] Formulations comprising a hydrogen peroxide source in combination with various metal halides were prepared and subjected to a variety of tests to assess their physicochemical properties and also, to ensure that the antimicrobial characteristics of the material had not been compromised in the reformulation activity. The ability of the material to form a stable pool of hydrogen peroxide was also assessed.

[0089] Microbial Strains:

[0090] Escherichia coli (NCIMB 8545), Staphylococcus aureus (NCIMB 9518) and Pseudomonas aeruginosa (NCIMB 8626) are grown on nutrient agar or in nutrient broth for 24 hrs at 37° C.

[0091] Candida albicans (NCIMB 3179) and Saccharomyces cerevisiae are grown on sabaroud dextrose agar or in sabaroud dextrose broth for 24 hrs at 37° C. sabaroud dextrose agar or in sabaroud dextrose broth for 24 hrs at 37° C.

[0092] Bacterial growth is monitored by measuring the culture optical density (OD) in a spectrophotometer (Anthos 2010) at a wavelength of 620 nm.

[0093] Antimicrobial Efficacy Improvement

[0094] Three formulations of A.sup.3IS were prepared by the addition of NaCl to result in the following concentration of NaCl to the aqueous phase before this is used to dissolve the sugars and enzyme.

TABLE-US-00003 NaCl (M) 0 0.5 1 A.sup.3IS (300 g) grmms grmms grmms Purified Water I 30 30 30 D-(−)-Fructose PhEur 114 110 105 D-(+)-Glucose PhEur 105 101 97 D-(+)-Maltose mono- 30 30 30 hydrate ≥95.0% Sucrose PhEur 4.5 4.5 4.5 Purified Water II 15 15 15 B-D-Glucose: 1.5 1.5 1.5 oxygen 1- oxidoreductase NaCl 0 8.77 17.53 Total 300 301 301

[0095] The formulations were assessed for antimicrobial activity against several species of micro-organisms (several of which produce catalase; Candida spp, Pseudomonas spp, E. coli) using the well diffusion bioassay, results were obtained by measuring the zones of inhibition (n=3).

[0096] Well Diffusion Methods—for Measurement of Microbial Inhibition

[0097] Agar plates are inoculated by swabbing overnight culture onto the plate surface. Plates are allowed to stand at room temperature for 15 minutes before use. Wells 8.2 mm diameter are bored into the surface of the agar. A 180 μl sample is placed into each well. The samples diffuse into the agar around the well and are assayed for an ability to produce a zone of inhibition. Plates are incubated for 24, 48 or 72 hrs and zones of inhibition are measured using an Autodata automatic zone reader. The diameter of zones, including the diameter of the well (8.2 mm), is recorded. HYDROGEN PEROXIDE CONCENTRATIONS AT TIME ZERO

TABLE-US-00004 H.sub.2O.sub.2 Concentration Formulation (mg/l) Range A.sup.3IS >0.5 < 100 A.sup.3IS + 0.5 M  >25 < 150 NaCl A.sup.3IS + 1.0 M  >25 <= 200 NaCl

[0098] The concentration of these materials in the final product will be in the following ranges:

TABLE-US-00005 Material Concentration Range NaCl 0.1-1.0 M

[0099] Antimicrobial activity tests were also carried out on NaCl with no discernible activity being found.

[0100] It can therefore be concluded that there is a synergistic action between A.sup.3IS and the ingredients added from an antimicrobial activity standpoint.

[0101] The new improved formulation gives rise to a higher concentration of hydrogen peroxide via sustained release.

Example 2: Effect of NaCl with A3IS

[0102] The composition comprising a metal halide (NaCl), hydrogen peroxide (A3IS) and a means for producing hydrogen peroxide (A3IS) was prepared according to the above-mentioned methods.

[0103] Different microbial strains were each plated out according to the above-mentioned methods.

[0104] The broad antimicrobial efficacy of this composition was then tested against each microbial strain according to the above-mentioned methods.

[0105] The results of this testing is shown in FIG. 3 and FIG. 4 and discussed above. A comparison with a control formulation is shown in FIG. 4 and also discussed above. The control is A3IS alone.

[0106] From the results it can be concluded that a synergistic action between A.sup.3IS and NaCl occurs that leads to an improved antimicrobial effect. This antimicrobial effect is shown across a broad range of microorganisms.

[0107] Formulation

[0108] The system of the present invention may be in many different physical forms, including but not limited to liquid preparations, solid or semi-solid preparations. In order to prepare solid or semi-solid formulations, the ingredients of the system should be manipulated to lower the water content and increase the content of the other components.

[0109] The system of the present invention may be in the form of a liquid preparation. Liquid preparations include but are not limited to a syrup, paste, spray, drop, ointments, creams, lotions, oils, liniments and/or gels. A typical gel includes an alcoholic gel such as isopropanol, ethanol, or propanol and/or a hydrogel.

[0110] Alternatively, the system of the present invention may be in the form of a solid or semi-solid preparation. Solid or semi-solid preparations include but are not limited to capsules, pellets, gel caps, hydrogels, pills, pillules and/or globules. Other means used for conventional drug-delivery can be adopted, for example, liposomal delivery may be contemplated.

[0111] According to a preferred embodiment of this aspect of the invention, there is provided a pharmaceutical composition comprising the system of the invention together with at least one pharmaceutically acceptable excipient or adjuvant.

[0112] According to another embodiment, there is provided a dressing comprising the system or pharmaceutical composition of the invention. Such dressings include gauzes, bandages, films, gels, foams—Lyofoam®, hydrocolloids—Granuflex®, alginates—Kaltostat® (Comvita), hydrogels—Intrasite Gel® and polysaccharide pastes, granules and beads.

[0113] According to a particular embodiment, the system may be present together with a wound-dressing matrix. Ideally, the ratio of the system to wound-dressing matrix may be approximately 1:1, although other ratios are contemplated. The wound-dressing matrix may be a collagen or collagen-GAG (glycosaminoglycan) matrix.

[0114] It will be understood that the system or pharmaceutical composition of the invention, may be present in many different adminstration forms. These forms include but are not limited to forms adapted for topical, enteral or parenteral administration.

[0115] Forms suitable for topical administration include a topical ointment, cream, lotion, oil, liniment, liquid and/or gel. For example, the system of the present invention may be applied epicutaneously, intranasally, via eye and/or ear drops. One particular embodiment of this aspect of the invention provides the system or pharmaceutical composition of the invention in a form adapted for intramammary administration. In this situation, the system or pharmaceutical composition of the invention may be adapted for delivery as part of a teat seal or intramammary depot delivered via the teat canal. Further compositions may be adapted as tissues, bandages or dressings. This is particularly advantageous for the treatment of infections such as mastitis and has both medical and veterinary applications.

[0116] Another form suitable for topical administration includes the system or pharmaceutical composition of the invention wherein the system or composition is in a form adapted for delivery via a dissolvable film strip or strips. In this situation the system of the present invention is soluble upon application.

[0117] Enteral administration includes, but is not limited to oral administration. Other enteral administration forms include suppositories and enemas. Forms suitable for oral administration include a capsule, pellet, gel cap, pill, pillule, globule, lozenge, dental floss, toothpaste, mouthwash, dissolvable film strips and/or adapted for delivery as part of a mouth guard. According to one embodiment of this aspect, the system or pharmaceutical composition is in a form suitable for controlled or sustained-release delivery. For example, the oral administration form may have an enteric coating to provide for controlled or sustained-release delivery. This sustained release aspect is important for the treatment of Campylobacter infections in poultry and the treatment of Cryptosporidium infections in cattle.

[0118] Parenteral/enteral administration forms include, but are not limited to injection. For example, the system may be adapted for injection by intramammary administration. This is particularly useful for the treatment of mastitis. Intramammary injection by this means involves injection directly into the teat canal using a tube or syringe with a nozzle of appropriate size, e.g. approx. 1.0 mm. Injection in this situation is directed into a body cavity or abscess.

[0119] The composition of the invention which includes 8 methoxy psoralen may be useful in treating psoriatic nail. The inclusion of this material together with exposure of the nails to UV-A light would be particularly beneficial.

[0120] Sodium chloride acts against catalyse which is a component in both vaginal fluid and semen so the composition of the invention can be used as a medicament in the treatment of bacterial vaginosis and in the preparation of a medicated condom.

[0121] The terms “comprise” and “include”, and any variations thereof required for grammatical reasons, are to be considered as interchangeable and accorded the widest possible interpretation.

[0122] The term “hydrogen peroxide source” will be understood to cover hydrogen peroxide itself and/or a means for generating hydrogen peroxide.

[0123] In the specification, it will be understood that the term “antimicrobial” or “antibacterial” are used interchangeably herein and cover biocidal or biostatic activity against various types of micro-organisms including but not limited to bacteria, fungi, viruses, yeasts, parasitic or pathogenic micro-organisms and/or moulds.

[0124] In the specification the term “by weight”, “percentage by weight” or “% w/w” refers to the weight of the final composition or system. These w/w values are interchangeable with w/v.

[0125] It will be understood that the components shown in any of the drawings are not necessarily drawn to scale, and, like parts shown in several drawings are designated the same reference numerals.

[0126] It will be further understood that features from any of the embodiments may be combined with alternative described embodiments, even if such a combination is not explicitly recited hereinbefore but would be understood to be technically feasible by the person skilled in the art.

[0127] The invention is not limited to the embodiments hereinbefore described which may be varied in both construction and detail within the scope of the appended claims.