ANTIMICROBIAL COMPOSITIONS AND METHODS FOR THEIR PRODUCTION

Abstract

This invention relates to a method for preparing compositions for preventing or treating microbial infections, compositions suitable for use in such treatments and methods for treatment or prevention of infections. One such composition finds particular use in treating mastitis in ruminants. The composition is administered into the udder of an animal as a highly effective treatment for mastitis, or as a prophylactic therapy, by means of an intra-mammary infusion. The milk produced by the animal, during treatment using the composition and method of the invention, is free of residues, such as antibiotics, antimicrobial agents or antimicrobial proteins, which could affect its suitability for drinking or in the production of milk products, such as cheese or yoghurt. The compositions and methods are also useful in treating and preventing lung infections; and infections in burns and wounds; and other infections caused by biofilms. The compositions may also be used on medical devices to prevent infection.

Claims

1. A method of treating or preventing mastitis in ruminants comprising: administering a pharmaceutical composition comprising iodide (I) and a source of hydrogen peroxide, together with a pharmaceutically effective carrier or diluent where the pharmaceutical composition does not include a peroxidase enzyme.

2. The method of claim 1, where the concentration of hydrogen peroxide in the pharmaceutical composition is at least one of the group consisting of less than 1% based on weight/volume of the pharmaceutical composition and less than 1% based on weight/weight of the pharmaceutical composition, and the concentration refers to the concentration during use treating or preventing mastitis in ruminants.

3. The method of claim 1, wherein a ratio by weight of iodide to hydrogen peroxide is a 0.2:1 to 3:1 ratio.

4. The method of claim 1, wherein a ratio by weight of iodide to hydrogen peroxide is a 0.38:1 to 1.52:1 ratio.

5. The method of claim 1, wherein the pharmaceutical composition further comprises 5-5,000 mg of iodide, prepared at a ratio of between 0.38:1 and 1.52:1 by weight of iodide to hydrogen peroxide.

6. The method of claim 1, wherein the pharmaceutical composition comprises 5-5,000 mg of iodide, prepared at a ratio of 0.76:1 by weight of iodide to hydrogen peroxide.

7. The method of claim 1, wherein the iodide is potassium iodide.

8. The method of claim 1, wherein the source of hydrogen peroxide is a hydrogen peroxide-urea adduct.

9. The method of claim 1, wherein the source of hydrogen peroxide is a peroxide-releasing percarbonate or a slow-releasing form.

10. The method of claim 1, wherein the pharmaceutical composition further comprises lactoferrin or glucocorticoid.

11. The method of claim 10, wherein the glucocorticoid is predinisolone, prednisone, or hydrocortisone.

12. The method of claim 1, wherein the peroxide and iodide components are delivered concurrently.

13. The method of claim 1, wherein the pharmaceutically effective carrier is an emulsion.

14. The method of claim 1, wherein the composition is adapted for intra-mammary delivery.

15. The method of claim 1, wherein the source of hydrogen peroxide is a hydrogen peroxide-urea adduct and the iodide is potassium iodide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIGS. 1a and 1b Artificial udder model photograph and schematic. Milk was fed through the top opening by means of a peristaltic pump. Doses were infused and samples were withdrawn by means of the teat canal; situated at the bottom.

[0071] FIG. 2 Modified Robbins device, replete with 12 polyurethane coupons to allow attachment and proliferation of the biofilm culture. The device was provided with influent media and irrigated, through the openings at both ends.

[0072] FIG. 3 Somatic Cell Count (initially, and 21 days after commencement of treatment) of 11 animals treated for two days, twice a day, with a formulation containing 100 mg potassium iodide and 100 mg hydrogen peroxide.

EXAMPLES

[0073] 1 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; comprised of hydrogen peroxide and iodide (a range of 50-1,000 mg of hydrogen peroxide and a range of 35-700 mg of potassium iodide).

[0074] 2 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide by weight (76 mg iodide and 100 mg of hydrogen peroxide).

[0075] 3 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (50 mg of hydrogen peroxide and 38 mg iodideprovided by 50 mg of potassium iodide or 45 mg sodium iodide).

[0076] 4 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (150 mg of hydrogen peroxide and 114 mg iodideprovided by 150 mg of potassium iodide or 135 mg sodium iodide).

[0077] 5 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (200 mg of hydrogen peroxide and 152 mg iodideprovided by 200 mg of potassium iodide or 180 mg sodium iodide).

[0078] 6 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (300 mg of hydrogen peroxide and 228 mg iodideprovided by 300 mg of potassium iodide or 270 mg sodium iodide).

[0079] 7 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (400 mg of hydrogen peroxide and 304 mg iodideprovided by 400 mg of potassium iodide or 360 mg sodium iodide).

[0080] 8 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (500 mg of hydrogen peroxide and 380 mg iodideprovided by 500 mg of potassium iodide or 450 mg sodium iodide).

[0081] 9 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (1,000 mg of hydrogen peroxide and 760 mg iodideprovided by 1,000 mg of potassium iodide or 900 mg of sodium iodide).

[0082] 10 The above compositions (1-9) where the peroxide is present as a peroxide releasing compound such as percarbonates or perhydrates. In such embodiments, the appropriate concentration of percarbonate can be calculated by the available peroxide content (typically 20-30%) of the percarbonate and the required concentration of peroxide. For example, 1000 mg of sodium percarbonate with 30% available hydrogen peroxide would release 300 mg hydrogen peroxide on dissolving in the environment. Similarly the use of 333 mg percarbonate would release 100 mg hydrogen peroxide.

[0083] 11 A composition used to produce a general antibacterial wash, used for example as a teat dip wash, containing freshly mixed source of hydrogen peroxide and iodide at a range of 10-10,000 mg hydrogen peroxide and 7.6-7,600 mg iodide.

[0084] 12 A composition used a general antibacterial wash, for example as a teat dip wash, containing freshly mixed source of ionic iodide wherein 19-7,600 mg iodide per litre is present (provided by 25-10,000 mg potassium iodide or 22.5-9,000 mg sodium iodide), and prepared at a ratio of between 0.38-1.52:1 of iodide to hydrogen peroxide.

[0085] 13 A composition used a general antibacterial wash, for example as a teat dip wash, containing freshly mixed source of hydrogen peroxide and iodide wherein 100 mg hydrogen peroxide per litre is present and 76 mg iodide per litre is present (provided by 100 mg potassium iodide or 90 mg sodium iodide).

[0086] 14 A composition used a general antibacterial wash, for example as a teat dip wash, containing freshly mixed source of hydrogen peroxide and iodide wherein 200 mg hydrogen peroxide per litre is present and 152 mg iodide per litre is presentprovided by 200 mg potassium iodide or 180 mg sodium iodide.

[0087] 15 A poultice prepared for wound care where the poultice is dipped into the antibacterial wash described in examples above (12-14).

[0088] 16 A compoisition as described above where the source of hydrogen peroxide is additionally, or alternatively, be provided by a peroxide releasing compound, such as percarbonate, or by enzymatic means, e.g. by the reduction of sugar compounds, sufficient to produce the required concentration of hydrogen peroxide. Furthermore, compositions that include an alternative appropriate oxidising compound to start the reaction, such as potassium permanganate, are included.

[0089] The source of iodide could additionally, or alternatively, be provided by a list of compounds set out elsewhere in this text, including, but not limited to sodium iodide, potassium iodide, lithium iodide, caesium iodide, hydrogen iodide, rhodium iodide, or other iodide releasing compounds, or a slow-releasing form of iodide, such as a degraded iodate.

[0090] Furthermore, iodide could be replaced by a thiocyanate molecule (supplied in the form of but not being limited to, for example, sodium or potassium thiocyanate) whilst adhering to a ratio of 0.2-5:1 or a more preferably 0.8-1.3:1 of thiocyanate to hydrogen peroxide) in areas where iodide use may pose an issue, for example, in people with thyroid problems.

[0091] This preparation can be administered by the use of consecutive suitable intra-mammary syringes, or a dual-barreled syringe (preventing premature reaction of iodide and peroxide).

[0092] All described compositions are to be prepared in a pharmaceutically suitable carrier. Such carriers include, but are not limited to, water, a pH suitable saline, a pH suitable saline including bicarbonate buffer, gel, or hydrogel.

[0093] Glucocorticoids from a list including, but not limited to prednisone, prednisilone, cortisol and hydrocortisone could be supplemented to the composition to aid with local inflammation issues during infection. For example, supplementation of 10-20 mg prednisolone or prednisone or 30-60 mg hydrocortisone to the antimicrobial composition could be used to counteract infection inflammation as part of a mastitis treatment.

[0094] The application of the composition will dictate the use of carrier. For example, mastitis intra-mammary treatment could be prepared in a saline or water, or in a more complex solution such as stearate/oil. A stearate/oil-based composition would be suitable in compositions to include the hydrogen peroxide-releasing sodium percarbonate as it prevents release of peroxide until the composition is diluted in milk in the mammary gland.

[0095] Furthermore, a hydrogel-based composition could be used to aid application in a wound care/bandage setting. Such an example is a sodium polyacrylate based hydrogel with a pH adjusted composition. Moreover, a saline or bicarbonate/saline carrier could be used in a disinfectant setting or for use as a nasal rinse to relieve sinusitis and accompanying infections.

[0096] In addition, the use of a temperature sensitive hydrogel containing poultice could be used to sequester the components such that application of the poultice to tissue at body temperature would allow the release of antimicrobial activity as the components react.

Pharmacological Results

Result 1

[0097] A freshly prepared mixture of hydrogen peroxide and potassium iodide was mixed (100 mg of each, at a ratio of iodide to hydrogen peroxide of 0.76:1 by weight) in 10 ml sterile water. Using a micro-broth dilution technique, the composition was doubly diluted in broth containing the test bacterial organism using a 96 well plate system. Depending on the sensitivity of the organism, a cut-off point was reached at a dilution at which the bacterial culture was no longer able to resist the effects of the antimicrobial composition. This is characterised as the minimum inhibitory concentration (MIC). The lower the MIC, the more sensitive the organism.

TABLE-US-00001 TABLE 1 MIC of mastitis causing organisms to an antimicrobial composition con- taining 100 mg hydrogen peroxide and 100 mg potassium iodide prepared freshly in a 10 ml volume and diluted accordingly. P. aeruginosa PA-29 exhibited increased tolerance to biocides and resistance to fluoroquinolone classed antibiotics. P. aeruginosa R strains are cystic fibrosis clinical isolates that exhibited tolerances one or more of amakcin, tobramycin, ciprofloxacin, and gentamicin. S. aureus BH1CC was described elsewhere as an MRSA class organism. MIC (mg/L of available Strain iodide/peroxide Escherichia coli ATCC 25922 20-40 Streptococcus dysgalactiae 143 (mastitis isolate) 5-10 Streptococcus dysgalactiae 160 (mastitis isolate) 5-10 Streptococcus uberis (mastitis isolate) 5-10 Staphylococcus aureus 15676 (mastitis isolate) 20-40 Staphylococcus aureus BH1CC (Rudkin et al., 2012) 20-40 Non-haemolytic coliform (mastitis isolate) 20-40 P. aeruginosa PA01 (wild type) 25-50 P. aeruginosa R550/2012 9026 (resistant CF isolate) 25-50 P. aeruginosa R468/2012 9027 (resistant CF isolate) 25-50 P. aeruginosa R479/2012 9028 (resistant CF isolate) 25-50 P. aeruginosa R480/2012 9029 (resistant CF isolate) 25-50 P. aeruginosa PA-29 (McCay et al., 2010) 25-50 Candida albicans 20-60 Candida tropicalis 20-60 Candida glabrata 20-60 Candida krusei 20-60 Saccharomyces cerevisiae 20-60

[0098] Table 1 shows the sensitivity of a number of mastitis-causing organisms to antimicrobial activity produced as a result of the reaction between hydrogen peroxide and iodide in the absence of a peroxidase enzyme. As is evident, all strains tested, including the Gram-positive organisms, were sensitive to the composition. It is evident that, in broth, the use of lactoperoxidase is not required. In addition, it is evident that the composition is also effective against fungi.

[0099] Furthermore, the broths were sub-cultured at various times post testing to assay for cell viability. This was carried out by sub-culturing 100 microlitres of the treated culture to 10 ml of fresh broth containing no antimicrobial. This differentiated between cell death and cell stasis (where viable cells would be detected). No growth was observed after 48, 72 or 96 hours post treatment (using concentrations in the same order of magnitude as those inhibiting them in the MIC test). This demonstrates that the produced antimicrobial activity is bacteriocidal to both Gram-negative and Gram-positive organisms, a surprising finding.

Result 2

[0100] The activity of the antimicrobial composition of the invention (peroxide and iodide) and another composition containing peroxide and thiocyanate against bacterial cells growing in biofilm mode was established by means of a modified Robbin's device (MRD). A biofilm of E. coli, S. aureus, P. aeruginosa, or a mixture of all three was established by inoculating 500 ml LB in a 1 L glass bottle that was connected to a modified Robbins device. The MRD system consists of 12 sampling ports, to which medical-grade polyurethane coupons (50 mm.sup.2 surface area) were inserted. The culture was allowed to proliferate at 37 C. and the MRD was fed/irrigated with culture at a rate of 0.1 h.sup.1 for at least 48 hours (a further 100 ml LB were supplemented after the initial 24 hours). The MRD was then irrigated with a control or a test solution. A small amount of constant force was used in the irrigation, such that it took approximately 90 seconds for the complete volume to pass through the system. The control used was a physiological saline solution. A number of distinct solutions were tested by irrigating the MRD chamber whilst the coupons were in situ: a 1 solution containing 0.3 g I.sup.1 potassium thiocyanate and/or potassium iodide, and 0.003% hydrogen peroxide; a 5 solution containing 1.5 g I.sup.1 potassium thiocyanate and/or potassium iodide, and 0.015% hydrogen peroxide; and a 20 solution containing 6 g I.sup.1 potassium thiocyanate and/or potassium iodide, and 0.06% hydrogen peroxide.

TABLE-US-00002 TABLE 2 Total viable counts (colony forming units per coupon) isolated from irrigated/non-irrigated E. coli biofilm and tested against a thiocyanate-based solution. Values represent the range from four coupons. Escherichia coli 1x 5x 20x No irrigation 10.sup.5-6 10.sup.5-6 10.sup.5-6 Saline 10.sup.5-6 10.sup.5-6 10.sup.5-6 Saline + SCN/H.sub.20.sub.2 10.sup.5-6 10.sup.5-6 10.sup.4-5

[0101] From Table 2 it is clear that 10.sup.5-6 colony forming units (CFU) were recoverable from the coupons in the absence of an irrigation step. Likewise, the bacterial viability was not significantly altered when the system was irrigated with a saline solution. The supplementation of thiocyanate and hydrogen peroxide to the solution at either 1 or 5 levels did not alter the outcome in cell viability. The use of a 20 solution did begin to affect cell viability, with a log-reduction in numbers typically noted. Similar results were noted when repeated with P. aeruginosa and S. aureus biofilm cultures).

TABLE-US-00003 TABLE 3 Total viable counts (colony forming units per coupon) isolated from irrigated/non-irrigated E. coli biofilm using an iodide-based solution. Values represent the mean from four replicates and two biological replicates. Escherichia coli 1x 5x 20x No irrigation 8.3 (0.8) 10.sup.5 2.2 (0.4) 10.sup.6 2.3 (0.8) 10.sup.6 Saline 8.2 (0.9) 10.sup.5 3.8 (0.2) 10.sup.6 1.9 (0.5) 10.sup.6 Saline + 5 (1) 10.sup.4 3.3 (0.5) 10.sup.3 NG Iodide/H.sub.20.sub.2 NGno viable cells recoverable.

[0102] The use of iodide instead of peroxide and thiocyanate derived OSCN.sup. had a profound effect on the outcome of the test (Table 3), against all three organisms. Cell viability was notably lower on use of a 1 iodide-based solution. Typical 100-1000-fold reductions in cell viability were noted after the 90 seconds irrigation (Table 3). A reduction from 10.sup.6 CFU to 10.sup.3 CFU was observed for E. coli on using the iodide at a 5 level. It should be noted that three of the four samples yielded no recoverable cells at all. A 20 solution was sufficient to result in a complete lack of viable cell recovery (Table 3).

[0103] Repetition of the set-up, but using a mixed culture of E. coli ATCC 25922, S. aureus DSM 15676 and P. aeruginosa NCIMB 10421 resulted in a very similar pattern (Table 4). A 1 solution was sufficient to result in a 100-fold reduction in bacterial viability. Both the 5 and 20 solutions were sufficient to result in a complete lack of viable cells recoverable from the coupon surface. Attachment to the surface was greater on using a mixed culture and there was some evidence to suggest that some cells were lost on irrigating with the saline solution only. Such a decrease in viability of biofilm attached cells within such a short space of time (90 seconds) would teach that the iodide/hydrogen peroxide composition would be very effective as a therapeutic in biofilm centred infections when involving an in vivo model.

TABLE-US-00004 TABLE 4 Total viable counts (colony forming units per coupon) isolated from irrigated/non-irrigated mixed biofilm of E. coli, P. aeruginosa, and S. aureus cultured together and tested against an activate-iodide solution. Values represent the mean from four replicates and two biological replicates. E. coli, Staph, P. aerug 1x 5x 20x No irrigation .sup.8.6 (1) 10.sup.6 .sup.7 (1.3) 10.sup.6 2 (0.4) 10.sup.7 Saline 2.6 (0.6) 10.sup.7 1.9 (0.4) 10.sup.6 5.9 (1.3) 0.sup.6 Saline + 5.9 (1.1) 10.sup.4 NG NG Iodide/H.sub.20.sub.2 NGno viable cells recoverable.

[0104] The data presented in Tables 3 and 4 would strongly indicate that the activity produced by even very low concentrations of the antimicrobial composition was capable of killing biofilm-formed bacterial cultures of both Gram-negative and Gram-positive organisms and even mixed culture consortia. This result would compare favourably to the use of antibiotics to achieve the same outcome. Many of the antibiotics would require 1,000-fold increases in concentration to achieve kills of biofilm cells by comparison to planktonic cells (i.e. free-floating). This was not the case when an iodide/hydrogen peroxide composition was employed to produce the antimicrobial activity.

[0105] We disclose that that hydrogen peroxide, even at low concentrations (e.g. a 0.003% solution), is capable of producing an effective antimicrobial action against infectious disease causing organisms, by reaction with iodide in the absence of a peroxidase enzyme. An increase in contact time (for example, 10 minutes) would allow 1 solutions to kill the bacterial cultures to the same degree noted here for stronger concentrations.

[0106] The prior art teaches that even higher concentrations of an antimicrobial agent will be required to effectively kill biofilm bacteria. The amount of hydrogen peroxide in the present invention is effective, however, when used with the correct amount of iodide. The composition of the invention will provide (a) a greater kill efficiency; (b) effective biofilm eradication and (c) a composition suitable for in vivo applications, such as treatment of mastitis or other infections (and doing so without causing irritation to the infection site).

Result 3

[0107] Lactoperoxidase has been shown to be present at significant concentrations in bovine milk, up to 30 mg I.sup.1. This level will depend on the health of the animal, breed of the cow etc. and therefore cannot be depended on to be present at optimal levels for an individual animal treatment. A reliance on naturally present lactoperoxidase would not allow an optimally designed treatment. To demonstrate that the described method and composition disclosed herein does not require endogenous lactoperoxidase, MICs were carried out in a broth, raw milk, pasteurised milk, and ultra heat treated (UHT) milk. The lactoperoxidase enzyme is inactivated at elevated temperatures, such as those used in the pasteurisation process. Typical pasteurisation processes (72 C. for 15 seconds) reduce activity of the enzyme by a significant amount (c. 70%), while treatment at 80 C. or more (including UHT treatment at 135 C.) would result in complete destruction of the enzyme. Were lactoperoxidase present in the milk to be essential to the reaction, the use of low concentrations of iodide and peroxide in a UHT milk environment should not produce any noted antimicrobial effect.

[0108] On testing the composition of the invention in these environments, E. coli was no more tolerant to the effects in UHT milk than that in broth, raw milk, and pasteurised milk (See Table 5). This clearly indicates that the composition of the invention is not in any way reliant on the presence of endogenous or native lactoperoxidase enzyme.

[0109] Furthermore, the supplementation of lactoperoxidase to the broths of UHT milk concentrations did not affect the outcome of the minimum inhibitory concentration determinations after 24 hours incubation (Table 5).

TABLE-US-00005 TABLE 5 MICs of E. coli ATCC 25922 to appropriately diluted formulation of 100 mg hydrogen peroxide and 100 mg potassium iodide. Values represent minimum mg/L value of potassium iodide and hydrogen peroxide required to inhibit growth over 24 hours. MIC (mg/L of available Test medium iodide/peroxide Water 20-40 LB broth 25-50 LB broth + 100 Units activity/LLP 25-50 LB broth + 500 Units activity/LLP 25-50 Raw Milk 25-50 Pasteurised Milk 25-50 UHT milk 25-50 UHT Milk + 200 Units activity/LLP 25-50 LBlysogeny broth. Pasteurised milk72 C. for 15 seconds. UHT milk135 C. for 3 seconds. LPlactoperoxidase.

Result 4

[0110] In an attempt to induce a tolerance to an antimicrobial, the micro-broth dilution method was utilised, with repeated passaging, with E. coli ATCC 25922 and a number of antibiotics, as well as the composition of the invention containing hydrogen peroxide and potassium iodide. Stock concentrations of kanamycin, polymyxin B, and levofloxacin (all clinically important and relevant antibiotics) were used to demonstrate the development of typical resistance characteristics to antibiotics. Doubling dilutions of antimicrobial compositions were made using a 96 well plate. The well contents were inoculated with 10.sup.4 cfu ml.sup.1 E. coli ATCC 25922 and allowed to incubate. The well with the highest concentration of antimicrobial that still resulted in growth overnight served as the inoculum for the next day's passage. Therefore, for example, the kanamycin-passaged strain did not come into contact with any of the antimicrobials during the test other than kanamycin.

[0111] After only 8 passages in the presence of the antibiotics, the organism was completely resistant to the kanamycin and polymyxin B drugs. After 10 passages, the organism was 8 times more tolerant to the effects of levofloxacin than it was pre-passaging. These results mirror the use of antibiotics in the environment wherein a bacterium will gain resistance due to repeated exposure to the antibiotics. However, during the same experiment, the organism was no more resistant to the effects of the antimicrobial activity produced by the hydrogen peroxide/iodide composition, after a similar number of passages. This is due to the inherent mode of action wherein there is no single target for the antimicrobial activity produced by this reaction, acting as is does across a variety of bacterial proteins. A bacterial cell would require multiple, concurrent, mutations to gain resistance, whilst only requiring one mutation in response to traditional antibiotics. This severely limits the ability of the organism to counter-act the inventions actions. Broad range acting antimicrobials have been described as unlikely to illicit bacterial resistance, or for development of cross-resistance toward antibiotics. The antimicrobial composition was also capable of killing a number of antibiotic-resistant P. aeruginosa bacteria. The developed kanamycin and polymyxin B resistant strains, as well as the levofloxain tolerant mutant, were no more tolerant to the actions of the disclosed composition, indicating the cross-resistance would not occur. A strain described previously by Mc Cay et al., (Microbiology, Vol. 156, No. 130-38, 2010) as being resistant to fluoroquinolone and tolerant to benzalkonium chloride biocide was sensitive to the actions of the antimicrobial activity of the invention (Table 1). Likewise, a number of clinical cystic fibrosis isolated P. aeruginosa strains, with elevated tolerances to key antibiotics used in the treatment of CF patients, were no more tolerant to the antimicrobial activity than the P. aeruginosa wild type strain (see Table 1). Further to this, S. aureus BH1CC (MRSA) is an antibiotic resistant strain (oxacillin MIC>256 mg/L) isolated from Beaumont hospital, Dublin, Ireland. The strain was no more tolerant to the actions of the reaction than another Staph. aureus strain (Table 1). This would again indicate that the method and composition disclosed here would be effective against MRSA isolates.

Result 5

[0112] An experimental method was devised to determine the most appropriate concentration of the composition required to provide antimicrobial action within the udder, but not persisting in the milk, post milking of the animal. A simulated bovine udder was designed, using an amended rubber gas-collection bag with two inlet channels (see FIG. 1). Various formulations, produced freshly by means of hydrogen peroxide and potassium iodide reactions, were introduced into the bag through an inlet. To simulate the udder environment, milk was then fed into the udder at the same rate a typical cow might produce milk, 12 L a day. Samples of the milk were taken routinely at various time points and the antimicrobial activity of the milk was determined by the spiking of the sample with E. coli. The spiked milk sample was then incubated and subsequently tested for the presence of remaining viable E. coli cells. A lack of viable cells indicated that there was sufficient antimicrobial activity present to inhibit/kill the E. coli. Conversely, the presence of viable cells indicated that the milk no longer contained sufficient quantities of antimicrobial activity to inhibit the growth of the bacterium. Using this method, each prospective embodiment was titred, such that activity was present for at least 5-6 hours post treatment/infusion, but no longer than 10 hours following treatment (such that activity will be absent when the milk is outside the udder environment). A 10 ml volume containing 100 mg potassium iodide, and 100 mg hydrogen peroxide achieved the desired characteristic (a 0.76:1 ratio of iodide to hydrogen peroxide by weight). At peroxide concentrations less than 50 mg (i.e. a >1.52:1 ratio of iodide to peroxide by weight), there was insufficient antimicrobial activity over a 4-5 hour period to be an effective treatment. At peroxide concentrations greater than 300 mg (a <0.38:1 ratio of iodide to peroxide by weight), antimicrobial activity persisted for greater than 10 hours, which could be a potential problem for post-processing of milk. The risk of damage to animal cell tissue also increases with increased peroxide concentrations. A ratio of between 0.38-1.52:1 of iodide to hydrogen peroxide by weight was suitable in providing a suitably paced reaction in the simulated cow udder.

[0113] Using this method, a suitable ratio of peroxide to iodide was predicted for the treatment various ruminant animals (cows, sheep, etc.). Interestingly, an undiluted 10 ml composition of peroxide and iodide (200 mg of each at a ratio of 0.76:1 iodide:hydrogen peroxide by weight) was found to have lost all significant antimicrobial activity within a smaller time frame than useful in a mastitis setting (though still useful for disinfectant purposes where contact time is much shorter), despite the elevated initial concentrations. This finding confirms that consideration of the kinetics of the reactions between hydrogen peroxide and iodide was a prerequisite for the development of suitable compositions for therapeutic purposes. It is counter-intuitive that a more concentrated solution of an antimicrobial compound would be less effective than a more dilute version and that the more concentrated solution would lose activity after a shorter period of time, by comparison to the embodiment containing 100 mg of each substancewhich again emphasises the link between reaction kinetics and efficacy in this case.

[0114] Failure to take into account the dynamic volume changes in the udder environment is also a problem with respect to producing an effective mastitis therapy. For example, a composition (100 mg potassium iodide and 50 mg hydrogen peroxidea 1.52:1 ratio by weight of iodide to hydrogen peroxide) was added to a constant, unchanging volume of milk of 1 L. The composition remained antimicrobially active for >24 hours in this situation, but the same composition recorded antimicrobial activity for <3 hours during tests in the udder model. Such a result, in the absence of an appropriate simulated udder model method, would teach that the antimicrobial composition was active for too long (i.e. greater than 10 hours) using this particular ratio and that activity would be expected long after the milk was taken from the udder. This proved that a simulated udder model, as disclosed in this application, provided an effective method, which allowed the design of an appropriate therapeutic for in vivo applications.

Result 6

[0115] The correct concentrations of peroxide and iodide are required to ensure an effective therapy for mastitis and to ensure that the post-processing of milk is not affected. The use of the model udder system allowed us to develop the correct concentration of composition. If an incorrect concentration is used the same results will not be observed.

[0116] For example, a lactating dairy cow was infused with a composition containing both potassium iodide and hydrogen peroxide (5 ml solution of each, containing 50 mg (converting to 38 mg iodide) and 150 mg, respectivelya ratio of 0.25:1 of iodide to peroxide by weight). A 5 ml embodiment was infused into lactating cow's udders, twice a day for 2 days. The animals were monitored for changes in activity, milk yield etc. Irritation, reduced milk yield and udder sensitivity was noted in the animals, indicating that the production of the antimicrobial activity was insufficient (or too slow) and that un-utilised peroxide was remaining, which started to irritate or damage the animal tissue. This experiment used a concentration of 0.1-0.15% peroxide in the udder, which although significantly lower than the concentrations of peroxide reported to be required in antiseptics, still resulted in irritation in the animals. A treatment for mastitis by use of such an embodiment would be unacceptable. Use of this embodiment (or a similar one where the peroxide is present for a prolonged period) would also lead to issues in post-processing of milk as well as initial irritation in the animal. This is because the presence of peroxide would likely inhibit any starter cultures used in the cheese and yogurt processing stages.

[0117] To demonstrate that the use of an alternative and preferred embodiment (containing 100 mg potassium iodide and 100 mg hydrogen peroxidea 0.76:1 ratio of iodide to peroxide by weight) would not harm animals, 5 lactating dairy cows were infused with this embodiment, twice a day, for 5 days. The animals were monitored for changes in activity, milk yield, milk quality, etc during the period immediately following treatment, and for several months after treatment. No signs of discomfort or changes in udder sensitivity, hardness or temperature were noted following treatment; and there was no change in the quality (nutritional, chemical or biological) or quantity of the milk produced by the individual animals. This demonstrated that: a) the composition and treatment was not toxic to the animal at the level described (even after a prolonged, 5 day treatment); b) there was no bottle neck in the reaction to produce antimicrobial activity resulting in a prolonged presence of peroxide; and c) that the likely cause of the irritation described earlier was the prolonged exposure of the teat canal tissue to excessive levels of peroxide. These specific results also support the suitability of this embodiment of the therapy for use as a treatment for sub-clinical mastitis and/or as a prophylactic therapy, given the absence of any negative effects on the cow and the possibility for continued utilisation of milk produced by an animal undergoing treatment.

Result 7

[0118] To demonstrate that the treatment method would not interfere with post-processing, milk taken from healthy animals, infused with a composition containing 100 mg potassium iodide and 100 mg hydrogen peroxide, was tested using a Delvo test. This is the gold standard for testing for the presence of antimicrobials (sensitive to parts per billion level for some antimicrobials and to a broad range of compounds). Milk produced from healthy cows and from cows with clinical mastitis, which were treated with the composition, recorded a negative result for antimicrobials when tested during treatment, thus proving that the antimicrobial composition was active for a limited period of time, i.e. between milkings only. Critically, this demonstrates the embodiment would not affect post-processing of the milkif there is insufficient antimicrobial activity present immediately post-milking to inhibit the Delvo test organism, then there is no issue with respect to the inhibition of starter cultures.

Result 8

[0119] A number of sick animals were identified that were suffering from chronic clinical mastitis due to S. aureus infections. These animals were non-responsive to previous antibiotic therapies in the months prior to the trial and demonstrated the typical cyclical SCC increases associated with chronic mastitis. Eleven of these animals were treated with an embodiment of the present invention, containing 100 mg potassium iodide and 100 mg hydrogen peroxide (0.76:1 by weight). The animals were treated twice a day, post milking, for two days. The somatic cell count was determined on day 21 post treatment start. This is in line with European Medicines Agency guideline dates (ensuring any effect noted is not merely transitory). At day 21, there was no evidence of any remaining clinical infection based on milk SCC counts, udder sensitivity, swelling, temperature or other milk quality indicators, thus demonstrating the therapeutic effectiveness of the method, even in cows chronically infected with S. aureus (see FIG. 3 for SCC count data). This trial demonstrates that the composition was capable of treating S. aureus infections of a chronic nature. Crucially, this also demonstrates that the composition was not harmful to the animals, as may be the case on using hydrogen peroxide. These animals were due to be culled (low milk yield, non response to numerous antibiotics, and continuous elevated SCC) but were returned to the herd and were milking normally for months following treatment using the invention. The estimated saving to the farmer due to increased milk production, no milk discard, and veterinarian fees, and replacement of animals was >10,000 ($13,000).

[0120] A novel finding from this in vivo study and the in vitro studies reported in this application using milk as bacterial growth mediumis the ability of the hydrogen peroxide/iodide model to function in vivo or in vitro in milk containing relatively elevated levels of thiocyanate. This would not be the case were a peroxidase enzyme employed to catalyse the reaction between hydrogen peroxide and iodide, based on the prior art.

[0121] Klebanoff et al., (J Exp Med 1967 126(6):1063-78) describe the fact that thiocyanate ions were inhibitory to the actions of IO.sup., produced by the peroxidase-catalysed reaction between hydrogen peroxide and iodide. The authors of that study believed this was an apparent paradox given the role of thiocyanate in the production of OSCN.sup.. Such a finding would further teach that the efficacy of the peroxidase-catalysed reaction between hydrogen peroxide and iodide to produce IO.sup. as an antimicrobial in the udder environment would be limited, as thiocyanate is present at significant concentrations in bovine milk (WHO/FAO, 2005). Tenovuo et al. and the articles discussed in that review (Oral Diseases {2002} 8, 23-29) stated that although an oxidation products of iodide are much more potent than thiocyanate oxidation products, the major problem with iodide in connection with LP-system is that even relatively small concentrations of salivary thiocyanate abolish the bacteriocidal effect of the LP/I/H.sub.2O.sub.2 systems as SCN is the preferred substrate. Moreover, it has been reported that a similar and strong interference between thiocyanate and iodide in an oral biofilm study of the yeast, Candida albicans strongly and negatively affects IO.sup. production. The authors state that the ability of OI.sup. to affect yeast growth and survival must be questioned in regard of the other iodine compounds, since clinical trials have shown limited or weak beneficial effects of other iodine formulations used for in vivo antifungal purposes. And The efficiency of an iodide/peroxidase system demonstrated in vitro through this investigation is difficult to transfer to either animal or human. Beside the toxicity of oxidant products on host cells and the immunogenicity of enzymes isolated for example from bovine milk, the oxidation of iodide is under the control of thiocyanate, which is not only present in several exocrine secretions (for example in human saliva) but is also preferentially used as substrate by lactoperoxidase. Indeed, simultaneous incorporation of both substrates in the same gel provided a decrease of the beneficial effect of 2 mM iodide in the presence of increasing concentrations of thiocyanate ranging from 0.25 to 4 mM, which correspond to the normal range of thiocyanate in saliva (Ahariz and Courtois 2010. Clinical, Cosmetic and Investigational Dentistry 2010:2 69-78). These authors conclude that peroxidase-generated IO.sup. to prevent C. albicans biofilm development would only be useful for treatment of devices in ex vivo conditions.

[0122] The concentrations of thiocyanate in milk are high, relative to the teaching described above, and they vary widely according to the animal diet. A severe inhibition of IO.sup. production by thiocyanate would thus suggest that a therapy based on iodide and hydrogen peroxide would be of no benefit for the treatment of mastitis.

[0123] The results from the described clinical trials of Result 8 carried out by the applicants recorded no negative impact of innate thiocyanate in milk on the efficacy of the proposed therapy, a highly surprising finding given the previous cited literature. It is explained by the need to balance the concentrations of the reactants appropriately, using the method described in the present invention, and of the critical importance of reaction kinetics to the antimicrobial efficacy of a therapy based on the antimicrobial activity produced by the reaction between peroxide and iodide.

Result 9

[0124] The simulated udder model method represents a significant advance on the prior art with respect to the assessment of antimicrobial activity. Results 6 and 8 above describe the use of the therapy with 100 mg potassium iodide and 100 mg hydrogen peroxide (0.76 iodide:1 hydrogen peroxide by weight). Use of too low a concentration of components resulted in no irritation or other negative effects to the animal, but also no significant drop in SCC counts or improvement in infection, during or post treatment. In vitro experiments using traditional minimum inhibitory concentration testing had, however, suggested that the solution would be antimicrobially active for c. 24 hours in a 1 L volume. The simulated udder model method correctly predicted, however, that it would not be antimicrobial for a sufficient period of time in vivo to result in a sufficient kill of the pathogen. The in vivo data validated the predictions of the experimental model and demonstrated its importance in mapping the kinetics of the udder environment. It is now evident that there is a range of concentrations and ratios that must be achieved in the reaction to be effective for use as a mastitis treatment, and that the described embodiment in Result 8 achieves this effective concentration for the treatment of bovine mastitis.

[0125] It is evident upon examining these results, that the described hydrogen peroxide/iodide composition is effective at killing a wide range of organisms (Table 1). It has been shown also that there was a significant difference in outcome between using this model, by comparison to one based on the production of OSCN.sup. with respect to treatment of biofilm cultures of both Gram-positive and Gram-negative organisms (Tables 2-4). The ratio of the compounds has also been shown to be extremely important at an in vivo level. Too high a concentration leads to tissue damage or prolonged activity (affecting post-processing of milk; failure of Delvo test). Too low a concentration or ratio leads to insufficient activity, minimising the effect of the treatment in killing the present pathogens. The kinetics of the udder environment, starting from a very low volume and increasing to 1-4 litres of milk is also an important factor. Surprisingly, undiluted compositions lost activity much quicker than diluted compositions. Traditional in vitro models using a constant final volume would teach one to over-estimate the effectiveness of the hydrogen peroxide/iodide model and would not translate to success in vivothis is a feature of the prior art cited in this application. The development, design and deployment of the novel simulated udder model method disclosed here allowed the proper evaluation of the disclosed treatment and ultimately enabled the development of the novel therapy for mastitis.

[0126] This is the first description of the reaction between low concentrations of hydrogen peroxide and iodide as being the basis for a highly efficient bacteriocidal composition against both Gram-negative and Gram-positive bacteria, independent of lactoperoxidase (or any other peroxidase enzyme) activity. It is the first to demonstrate that the even low concentrations of the reaction products of hydrogen peroxide and iodide provide an efficient means of eradicating bacterial biofilms, even in the presence of thiocyanate. It is also the first disclosure of the successful use of a hydrogen peroxide and iodide composition for therapeutic use for treatment of bovine mastitis, and in a potential open wound setting by way of a poultice.

[0127] This is the first description of an experimental method to characterise the evolving udder environment in order to allow the development of a mastitis therapy based on hydrogen peroxide and iodide. We present in vivo data to validate the efficacy of the novel method to produce a safe treatment for bovine mastitis, a method capable of both treating and curing mastitis, while simultaneously allowing producing of milk that will not affect the post-processing processes, such as cheese or yoghurt production.

Result 10

[0128] To demonstrate the difference between traditional iodine based antimicrobials and the activated IO.sup. form described herein, MICs were performed in a variety of media, including water, LB broth, and milk, using E. coli ATCC 25922 as a test organism. As a comparison, PVP-1 or povidone iodine, used frequently in hospital setting as a topical antiseptic, was used as a source of free iodine (12). Although the PVP-1 is strongly antimicrobial in water and saline, it decomposes extremely quickly in the presence of organic material present in LB and milk (see Table 6). This limits the efficacy of PVP-1 to such areas where organic material is not present. This is not the case with the present OI.sup. formations. Organic material does not irreversibly render the solution ineffective. Curiously, the use of a combined hydrogen peroxide and PVP-1 in a saline environment resulted in no antimicrobial activity either, as the PVP-I apparently exerts a negative effect on peroxide antimicrobial activity (Table 6). Repetition in a broth environment causes the PVP-1 to be broken down before it can affect the peroxide, and the MIC is identical to that of peroxide in the absence of PVP-1. Using the composition of 100 mg H.sub.2O.sub.2 and 100 mg KI, MICs of between 20-40 mg L-1 were achieved in each of the test environments (milk, broth and water), indicating that the presence of organic material did not inhibit the desired antimicrobial activity.

TABLE-US-00006 TABLE 6 Activity of and PVP-I in absence and presence of organic material Solution environment MIC (mg L.sup.1) PVP-I (saline) 1-2 PVP-I (in LB) >250 PVP-I (milk) >250 PVP-I + H.sub.20.sub.2 (saline) >250 PVP-I + H.sub.20.sub.2 (broth) 20-40 H.sub.2O.sub.2 (broth) 20-40