WO2013028082A1

WO2013028082A1 - Antimicrobial composition

Info

Publication number: WO2013028082A1
Application number: PCT/NZ2012/000136
Authority: WO
Inventors: Karen Yeritsyan
Original assignee: Donaghys Industries Limited
Priority date: 2011-08-05
Filing date: 2012-08-03
Publication date: 2013-02-28
Also published as: NZ594450A

Abstract

Anti-microbial compositions, methods and uses are described. The composition is a liquid including a therapeutically effective amount of at least one sulphonic acid compound; lactic acid; and at least one citrate compound. The inventor has identified that the combination confers a synergistic antimicrobial effect above the individual compounds alone or in combinations of two of the compounds. The combination may also reduce the rate of additional antimicrobial component used, assuming that additional agent is used. The composition is also biodegradable and physiologically acceptable to topical application to an animal.

Description

ANTIMICROBIAL COMPOSITION

RELATED APPLICATIONS

This application derives priority from New Zealand patent application number 594450 incorporated herein by reference.

TECHNICAL FIELD

The application relates to an antimicrobial composition. More specifically, an antimicrobial composition is described that contains a combination of agents that act synergistically to enhance the antimicrobial nature of the combination and which also confer further useful properties to the composition.

BACKGROUND ART

Antimicrobial compositions are widely used to reduce the risk of infection and prevention of infection. For example, antimicrobials are used to disinfect surfaces in hospitals, lavatories, food preparation facilities, and offices. Other uses include the control of pathogenic organisms on skin, where they may be used to reduce the transmission of disease or infection, for example, as surgical scrub solutions, hand sanitizers, etc. Antimicrobial compositions may also be used in veterinary applications for the control or prevention of hoof diseases, mastitis (in milk producing animals), or topical infections. Prevention of mastitis is a major goal of the dairy industry, where the disease may result from contact of the bovine or ovine mammary gland with pathogenic microorganisms, usually bacteria but occasionally yeast or fungi.

Mastitis is the single most costly disease affecting the dairy industry. Mastitis is always a potentially serious infection. Severe cases may cause death to the dairy animal. Milder cases are more common, but may have serious consequences, such as long-term damage to the animal, loss of milk production for the dairy farmer and an unacceptable increase in veterinary costs.

To reduce mastitis, commercial teat dips and sprays have been developed which are usually administered to the teat by dipping, foaming, or spraying the teat prior to milking as well as after removal of the milking cup. Based on time of application, these dips and sprays can be divided into two groups: pre-milking and post-milking. Teat dips applied subsequent to milking may be in the form of lower viscosity dips or sprayed compositions or in the form of a thick composition, film or barrier that remains on the teat until the next milking, which is generally 8 to 12 hours later.

Teat dips/sprays have used a variety of antimicrobial agents. For example US 2,739,922^" describes the use of polymeric N-vinyl pyrrolidine in combination with iodophors. US 3,993,777 describes the use of halogenated quaternary ammonium compounds. US 4,199,602 describes the use of iodophors, chlorine releasing compounds (e.g. alkali hypochlorite), oxidising compounds (e.g. hydrogen peroxide, peracids), protonated carboxylic acids (e.g. heptanoic, octanoic, nonanoic, decanoic, undecanoic acids), and nitroalkanols. US 4,434,181 describes the use of acid anionics (e.g. alkylaryl sulfonic acids), chlorine dioxide (from alkali chlorite), and bisbiguanides such as chlorhexidine.

The majority of marketed teat care products are based on chlorhexidine gluconate, iodophors and fatty acids and chlorine although teat dips/sprays do exist with other antimicrobial agents such as ammonium quaternary compounds and hydrogen peroxide.

Some teat care agents suffer from serious drawbacks. For example, iodine, hypochlorite, chlorine dioxide, and hypochlorous acid are powerful disinfectants and strong oxidants, but they are also particularly noxious for both humans and animals. Chlorhexidine, for example, has become the focus of regulatory concern. In additional, the use of overly powerful disinfectants may contribute to the mastitis problem by causing irritation of the teat skin, thus providing an opportunistic site that promotes infection. Furthermore, the iodine-based and chlorine-based compositions may induce sensitised reactions in cow teats. This issue is of particular importance for barrier type products where the biocide may remain in contact with the skin during the 8-12 hour inter-milking period. On the other hand, less powerful teat dip agents, such as fatty acids and anionic surfactants, are often not broad enough in their antimicrobial spectrum to provide complete germicidal protection.

There is also an issue of milk contamination. Many existing bactericides used as active ingredients in udder care products are foreign materials to that found normally in milk and they may leave an after taste in milk. For example, it is well known that relatively small quantities of iodine and chlorhexidine can result in taste changes of the milk as well as problems in the manufacture of dairy products. Furthermore, milk products must meet food and drug regulations that take into consideration ingestion of residual teat dip agents. There may be concern, for example, about increased iodine consumption because iodine is linked to thyroid function. Also, iodine associates with problems of staining, and some operators/users develop allergic symptoms such as skin irritation and sensitization from iodine-based product use. It is acknowledged that art exists describing one or two of the organic acids described herein used for anti-microbial formulations. It should be noted though that these acids are used as pH adjusters and not as active agents themselves. For example the organic acids noted in the art may be used to reduce the pH of the overall formulation while the active agent itself may be iodine or other agents. In addition, the prior art does not recognise any synergism from a combination of three organic acids as identified by the inventors. Further, art anti-microbial formulations are typically formulated for application to hard and inert surfaces such as steels or plastics. The formulations are not pharmaceutically and physiologically acceptable and, if they were applied to a skin surface for example, the affects on the skin would be damaging due to the low pH or other non-physiologically acceptable properties of the art formulations. Based on the above it should be appreciated that there is a need for compositions that are effective broad spectrum antimicrobials that provide extended germicidal activity and are non- irritating to the skin or at least to give the public a choice.

For the purpose of this specification the term 'comprise' and grammatical variations thereof shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements.

Further aspects and advantages of the process and product will become apparent from the ensuing description that is given by way of example only.

SUMMARY OF THE INVENTION

The application broadly relates to synergistic anti-microbial compositions including a combination of a sulphonic acid compound, lactic acid and a citrate compound. The combination confers a synergistic antimicrobial effect particularly against C. bovis above the individual compounds alone or in combinations of two of the compounds. The combination also reduces the amount of additional antimicrobial component used assuming that additional agent(s) are used.

In a first aspect there is provided a pharmaceutically and physiologically acceptable antimicrobial composition including a therapeutically effective amount of the active agents:

(a) at least one sulphonic acid; and

(b) lactic acid; and

(c) at least one citrate compound;

wherein the composition is a liquid formulated for topical application to a non-human animal.

In a second aspect there is provided a pharmaceutically and physiologically acceptable antimicrobial composition including 10-50% w/v of the total composition active agents, wherein the active agents include:

(a) 30-69% w/v of the total agent concentration dodecyl benzene sulphonic acid; and

(b) 30-50% w/v of the total agent concentration lactic acid; and

(c) 1-20% w/v of the total agent concentration sodium citrate; and

In a third aspect, there is provided a method of preventing and/or treating a microbial infection in a non-human animal by topical administration of a composition substantially as described above.

In a fourth aspect, there is provided a method of sterilising the udder region or regions of a lactating non-human animal by the step of topically applying the composition substantially as described above to the udder region or regions of the animal.

In a fifth aspect, there is provided a method of sterilising the skin of a non-human animal by topical administration to the non-human animal of a pharmaceutically and physiologically acceptable liquid composition including a therapeutically effective amount of the active agents:

(a) at least one sulphonic acid; and

(b) lactic acid; and

(c) at least one citrate compound.

In a sixth aspect, there is provided a method of sterilising a skin area from C. bow^'s infection by topical application to the skin area of a non-human animal, a pharmaceutically and

physiologically acceptable liquid composition including a therapeutically effective amount of the active agents:

(a) at least one sulphonic acid; and

(b) lactic acid; and

(c) at least one citrate compound.

In a seventh aspect, there is provided the use of a composition substantially as described above in the manufacture of a medicament for the treatment of a microbial infection in an animal.

Advantages of the above compositions, methods and uses include the fact that the composition is highly effective as a antimicrobial composition against many bacteria and is synergistically active against the bacteria Corynebacterium bovis. The composition also synergistically reduces the amount of any other antimicrobial agents added to the composition (if such extra compounds are desired). The composition also utilises biodegradable components unlike art formulations that require non-biodegradable compounds. A further advantage is that the composition is physiologically acceptable and hence suitable for direct topical administration without issues such as skin irritation or damage to the animal tissue that may be the case with other art anti-microbial formulations.

DETAILED DESCRIPTION

As noted above, the application broadly relates to synergistic anti-microbial compositions including a sulphonic acid compound, lactic acid and a citrate compound. The combination confers a synergistic antimicrobial effect above the individual compounds alone or in combinations of two of the compounds. The combination also reduces the rate of additional antimicrobial component used, assuming that additional agent is used.

For the purposes of this specification, the term 'therapeutically effective amount' or grammatical variation thereof is intended to qualify the amount of topical composition that will achieve the desired level of microbial kill. 'Therapeutically effective' may also refer to a decrease in frequency of infection or a decrease the severity of an infection.

The term 'sulphonic acids' or grammatical variations thereof refer to organic acid based compounds with a sulphonate group (-S0₃H).

The term 'topical' or grammatical variations thereof refer to application of the composition to the skin or mucous membranes of an animal. The terms 'teat dip' and 'teat spray' or grammatical variations thereof encompass application of the composition to the animal teats but may also encompass application to other parts of the udder as well encompassing other means of application such as painting and foaming.

The term 'about' or 'approximately' or grammatical variations thereof refer to a quantity, a level, a degree, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, a level, a degree, value, number, frequency, percentage, dimension, size, amount, weight or length.

The term 'substantially' or grammatical variations thereof refers to at least about 50%, for example 75%, 85%, 95% or 98%.

The term 'pharmaceutically and physiologically acceptable' or grammatical variations thereof refers to the composition when administered to an animal being both active as an anti-microbial and doing so in a manner that is in accord or characteristic of the normal functioning of the animal in a healthy state and does not cause skin irritation or other adverse skin reactions. In a first aspect there is provided a pharmaceutically and physiologically acceptable antimicrobial composition including a therapeutically effective amount of the active agents:

(a) at least one sulphonic acid; and

(b) lactic acid; and

(c) at least one citrate compound;

It was unexpectedly found by the inventor that a combination of a sulphonic acid compound or compounds, lactic acid and a citrate compound or compounds showed significantly increased antimicrobial properties when compared to the individual compounds alone or in combinations of two are used as antimicrobial agents. This synergism was particularly noticeable for the microbe C. bovis, a difficult and persistent bacterium that is hard to effectively sterilise using art formulations.

The combination of active compounds noted in (a) to (c) above may comprise about 10, or 15, or 20, or 25, or 30, or 35, or 40, or 45, or 50% w/v of the total composition. The combination may comprise about 20-30% w/v of the total composition.

The sulphonic acid compound may be one with a carbon number less than twenty. The sulphonic acid may be an anionic surfactant. The sulphonic acid compound may be dodecyl benzene sulphonic acid or salts thereof e.g. sodium or calcium salts. The sulphonic acid compound may be linear dodecyl benzene sulphonic acid. The dodecyl benzene sulphonic acid may be included at a rate of about 30, or 35, or 40, or 45, or 50, or 55, or 60, or 65, or 69% w/v of total active agents in the composition. The dodecyl benzene sulphonic acid may be included at a rate of about 40-64% w/v of total active agents in the composition.

Dodecyl benzene sulphonic acid (or linear alkyl benzene sulphonic acid, also known as lauryl benzene sulphonic acid) is one of the largest volume sold synthetic surfactants, because of its relatively low cost, good performance, the fact that it can be dried to a stable powder and the biodegradable environmental friendliness it has, particularly when in linear chain form. Dodecyl benzene sulphonic acid is an anionic surfactant with molecules characterised by a hydrophobic and a hydrophilic group.

As an anti-microbial agent, the inventor's understanding is that dodecyl benzene sulphonic acid acts to denature the proteins of microbial cells, inactivate essential enzyme systems, and disrupt cell membranes.

Lactic acid may be included at a rate of about 30, or 35, or 40, or 45, or 50% w/v total active agent in the composition. Lactic acid may be included at a rate of about 35-45% w/v total active agent in the composition.

Lactic acid is widely used in skin care. It is an alpha-hydroxy acid (AHA) and AHAs' have been used in cosmetic products intended for daily application to skin. The functional benefits provided by lactic acid are skin moisturising and exfoliating. For this reason it is formulated into skin creams and lotions to help mitigate the appearance of wrinkles and the signs of aging skin. Lactic acid is inhibitory to both gram-positive and gram-negative organisms. Lactic acid is a food grade chemical and is non-irritating to skin. Lactic acid does not leave harmful residues in milk and is tolerant to organic matter. In short, lactic acid is anti-microbial, naturally compatible with milk and confers skin-conditioning properties to compositions containing lactic acid.

The citrate compound may be a metal citrate salt. The metal citrate salt may be sodium citrate, calcium citrate or potassium citrate. Sodium citrate is useful as it is widely available and inexpensive but should not be seen as limiting as other citrate salts may also be used. The citrate compound or compounds may be included at a rate of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20% w/v of total active agent in the composition. The citrate compound or compounds may be included at a rate of about 1 -15% w/v of total active agent in the composition.

Sodium citrate is widely used in applications such as foods (e.g. in ice-cream production used to keep the fat globules from sticking together), as an anti-coagulant, in detergents and cleaner production (as a sequestering agent), in medicine (for the treatment of urinary track infections) and other applications.

The ratio of lactic acid to citrate compound or compounds in the composition may be about 6-10 parts lactic acid to about 1 -1.5 parts citrate compound or compounds. The inventor has found that this ratio may be important in attaining the desired synergies noted below.

The ratio of sulphonic acid compound or compounds to citrate compound or compounds may range from about 6 parts sulphonic acid to about 1 part citrate to about 10 parts sulphonic acid to about 1 part citrate.

The composition may include at least one carrier. The carrier may form about 40-80% w/w of the composition. The carrier may be water. The carrier may be deionized water. It should be appreciated that other solvents or compatible materials other than water may be used to achieve the effective concentrations of germicidal agents e.g. glycols such as monopropylene glycol, sugar alcohols such as sorbitol and polyols such as glycerine. In some embodiments, a composition may contain at least about 50%, or about 60% water by weight based on the total weight of the composition.

The composition may also contain at least one additive selected from: a buffering/pH adjusting agent, a film/barrier forming agent, a wetting agent / surfactant, a viscosity controlling agent, an opacifying agent or dye, a skin conditioning agent, a preservative, an emollient and

combinations thereof.

The additive may be at least one buffering and/or pH adjusting agent. A composition pH value may be selectively adjusted by the addition of acidic or basic ingredients. Suitable acids for use as pH-adjusting agents may be selected from: phosphoric acid, nitric acid, glycolic acid, and combinations thereof. Mineral acids may also be used to lower the pH. The pH may be raised or made more alkaline by addition of an alkaline agent selected from: sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, monosodium acid diphosphonate, and combinations thereof. A useful alkaline may be sodium hydroxide, due to sodium lactate formation, which by itself is known to be very effective skin conditioning agent.

Generally, an acidic pH composition may be preferable. The composition pH may range from about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0. The pH range may be about 3.0 to 5.0. The pH range may be about 2.5 to 4.5.

The additive may be at least one film/barrier-forming agent. The film/barrier forming agent may be added sufficient to be capable of forming a long-lasting, persistent, continuous, uniform barrier film. In one embodiment, the film/barrier forming agent may be added to a concentration of about 0.01 -20% by weight film forming agent.

The barrier and film forming agents may be selected by their ability to remain in contact with the teat between milking cycles. Barrier and film forming agents coat the teat skin and, optionally, the udder. Barrier agents may form a plug at the end of the open teat canal. Typical barrier and film forming agents may be selected from thick creams or emollients (made with viscosity control agents), films, polymers, latex and the like. In the inventor's experience, some non-ionic surfactants may help further enhance the barrier properties of a composition, in addition to contributing to surface wetting. Examples of such surfactants may include polyoxyethylene- polyoxypropylene glycol. Suitable barrier forming agents may be selected from: latex, arabinoxylanes, glucomannanes, guar gum, johannistree gums, cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, carboxyethyl cellulose,

carboxymethyl cellulose, starch, hydroxyethyl starch, gum arabic, curdlan, pullulan, dextran, polysulphonic acid, polyacryl amide, high molecular weight polyacrylate, high molecular weight cross-linked polyacrylate, carbomer, glycerol, sodium alginate, sodium alginate cross-linked with calcium salt, xanthan gum, polyvinyl alcohol) (PVA) and poly(N-vinylpyrrolidone) (PVP).

Barrier-forming agents that the inventor has found may be advantageous from this list include: xanthan gum, carboxymethyl cellulose, sodium alginate, sodium alginate cross-linked with calcium salt, PVA, hydroxyethyl cellulose, PVP, and (2,5-dioxo-4-imidazolidinyl)-urea (Allantoin).

The additive may be at least one wetting agent / surfactant. The wetting agent / surfactant may be added to a level of 0.2% w/v to 15% w/v. The wetting agent / surfactant may be added to a level of 0.5% w/v to 5% w/v. Wetting agent(s) or surface-active agent(s) are also known as surfactants. Typical wetting agents are used to wet the surface of application thereby reducing surface tension so that the agent(s) can penetrate the surface and to also remove any unwanted soil on the application surface. The wetting agents or surfactants of the composition increase overall detergency of the formula, solubilize or emulsify some of the organic ingredients that otherwise would not dissolve or emulsify, and facilitate penetration of active ingredients deep onto the surface of the intended application surfaces, such as teat skin.

Suitably effective surfactants used for wetting in the present composition may include anionic, cationic, non-ionic, amphoteric surfactants. Further, wetting agents and surfactants used in the present composition may be high foaming, low foaming and non-foaming types. Suitable anionic surfactants may be selected from: a linear alkyi benzene sulphonate, an alkyi a- sulphomethyl ester, an a-olefin sulphonate, an alcohol ether sulphate, an alkyi sulphate, an alkylsulpho succinate, a dialkylsulpho succinate, and alkali metal, alkaline earth metal, amine and ammonium salts thereof. Suitable non-ionic surfactants may be selected from: an alkyi polyglucoside, an alkyi ethoxylated alcohol, an alkyi propoxylated alcohol, an

ethoxylatedpropoxylated alcohol, sorbitan, sorbitan ester, and an alkanol amide. Amphoteric surfactants may be chosen from alkyi betaines and alkyi amphoacetates. Suitable betaines may include cocoamidopropyl betaine, and suitable amphoacetates may include sodium

cocoamphoacetate, sodium lauroamphoacetate and sodium cocoamphodiacetate. It should be understood that where at least one surfactant/emollient (e.g. alkyi ethoxylated alcohol) is included, that these surfactants would also have an influence on the wetting properties of the mixture.

The additive may be at least one viscosity-controlling agent.

The term 'liquid' or grammatical variations thereof when used in respect of the composition described above refers to a dynamic viscosity ranging from about 1 -3000cPs. While this is a wide range, it should be appreciated that the breadth of application methods means a wide range of viscosity is likely e.g. an ointment versus a liquid spray.

As noted above, viscosity control agents may be added to formulate the antimicrobial compositions according to an intended environment of use. It may be advantageous for some compositions to have an optimised solution viscosity to impart vertical clinging of the product onto a teat. This type of viscous product, especially one having a suitable thixotropic, pseudoplastic or viscoelastic gel strength, minimises dripping of the product to avoid wastage and is particularly advantageous in teat dip compositions. Suitable viscosity control agents may be selected from: hemicellulose, for example arabinoxylanes and glucomannanes; plant gum materials, for example guar gum and johannistree gums; cellulose and derivatives thereof, for example methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose or carboxymethyl cellulose; starch and starch derivatives, for example hydroxyethyl starch or cross linked starch; microbial polysaccharides, for example xanthan gum, sea weed polysaccharides, for example sodium alginate, carrageenan, curdlan, pullulan or dextran, dextran sulphate, whey, gelatin, chitosan, chitosan derivatives, polysulphonic acids and their salts, polyacrylamide, and glycerol. Advantageous viscosity adjusting agents in the inventor's experience may include different types of cellulose and derivatives thereof such as hydroxyalkyl cellulose,

methylcellulose, and glycerol. Typical viscosity control ingredients may be selected from:

xanthan gum, carboxymethyl cellulose, sodium alginate, sodium alginate cross-linked with calcium salt, polysulphonic acids and their salts, polyacrylamide, polyvinyl alcohol (PVA), hydroxyethyl cellulose and poly-N-vinylpyrrolidone) (PVP).

The additive may be at least one opacifying agent or dye. An opacifying agent or dye may optionally be included in the present composition. A colour may be helpful, for example, colour on a teat tells a farmer that a particular cow has been treated. To preclude any problems with possible contamination of milk, it may be preferable to use only FD&C Certified (food grade) dyes. There are many FD&C dyes available including for example: FD&C Red #40, FD&C Yellow #6, FD&C Yellow #5, FD&C Green #3 and FD&C Blue #1. Dyes may be used either alone or in combination. D&C Orange #4 may also be used. Titanium dioxide (Ti0₂) is widely used as an opacifier and may also be used alone or in combination with various colorants. Other possible dyes that may be used include Carmosene Red and Acid Green.

The additive may be at least one skin-conditioning agent. The skin-conditioning agent may be included at a rate of 5-30% w/v. The skin-conditioning agent may be included at a rate of 8-20% w/v. Skin conditioning agents may provide extra protection for the skin prior to or subsequent to being exposed to a challenge. For example, skin conditioning agents may include moisturisers, such as glycerin, sorbitol, propylene glycol, lanolin, D-panthenol, polyethylene glycol (PEG) 200- 10,000, polyethylene glycol esters, acyl lactylates, polyquaternium-7, glycerol cocoate/laurate, PEG-7 glycerol cocoate, stearic acid, hydrolysed silk peptide, silk protein, aloe vera gel, guar hydroxypropyltrimonium chloride, alkyl poly glucoside/glyceryl laurate, shea butter and coco butter; sunscreen agents, such as titanium dioxide, zinc oxide, octyl methoxycinnamate (OMC), 4-methylbenzylidene camphor (4-MBC), oxybenzone and homosaiate; and itch-relief or numbing agents, such as aloe vera, calamine, mint, menthol, camphor, antihistamines, corticosteroids, benzocaine and paroxamine HCI.

The additive may be at least one preservative. The preservative may be included at a rate of about 0.1-1 % w/v. The preservative may be ethylenediaminetetraacetic acid (EDTA) and/or its alkali salts which can act as a chelating agent to remove metal ions from hard water. Metal ions, if not removed from the composition, may serve as reaction sites for enzymes within the bacteria with metalloenzyme reactions producing energy for bacterial cell replication. Other

preservatives may also be used selected from: paraban, methyl paraban, ethyl paraban and glutaraldehyde. Preservatives such as an alcohol may also be added. The alcohol used may_ be benzyl alcohol, a low molecular weight alcohol having a carbon number less than five, and combinations thereof.

The additive may be at least one emollient. The emollient may be included at a rate of about 5- 30% w/v. The emollient may be included at a rate of about 8-20% w/v. It should be appreciated that the emollient may be lactic acid itself hence no further separate emollient may be added. Alternatively, additional emollient may be added selected from compounds including glycerine and/or lanolin. An aim of using an emollient in addition to lactic acid may be to prevent skin cracking and irritation.

Additional antimicrobial agents may be added to the composition although are not essential to result in a sterilising effect. The inventor unexpectedly found that the present composition significantly or synergistically reduces the dose amount of additional antimicrobial agent required in order to achieve the same effect as the additional antimicrobial agent alone, one example being a reduced requirement for iodine.

Anti-microbial agents are the components of a composition that destroy microorganisms or prevent or inhibit their replication. The present composition may be used in combination with other antimicrobial agents. This may be advantageous, for example, to achieve an effective kill at lower concentrations of traditional antimicrobial agents. The present composition may also be advantageous to use with other antimicrobial agents in order to maintain good animal skin condition by preventing skin irritation, which may occur when "aggressive' antimicrobial products are used. As noted above, the inventor has found that a lower concentration of antimicrobial agent is required when used with the present composition. As a result, this softens the negative effect of the additional antimicrobial agent on skin. A reduction in concentration of other antimicrobial agents also minimises possible contamination of milk product with the extra antimicrobial agent. Additional antimicrobial agents may be selected from: iodophors, quaternary ammonium compounds, hypochlorite releasing compounds (e.g. alkali hypochlorite, hypochlorous acid), oxidizing compounds (e.g. peracids and hypochlorite), protonated carboxylic acids (e.g. heptanoic, octanoic, nonanoic, decanoic, undecanoic acids), chlorine dioxide from alkali chlorite by an acid activator, and bisbiguanides such as chlorhexidine. Phenolic antimicrobial agents may be chosen from 2,4,4'-trichloro-2"-hydroxydiphenylether. Another such antimicrobial agent is 4-chloro-3,5-dimethyl phenol. Other traditional germicides include formaldehyde releasing compounds such as glutaraldehyde and 2-bromo-2-nitro-1 ,3- propanediol, polyhexamethyl biguanide, guanidine salts such as polyhexamethylene guanidine hydrochloride, polyhexamethylene guanidine hydrophosphate and poly[2-(2-ethoxy)- ethoxyethyl]-guanidinium chloride and mixtures thereof. In one embodiment, the composition may be used in combination with traditional germicides such as copper sulphate, zinc sulphate, sulphamethazine, quaternary ammonium compounds, hydrogen peroxide and/or peracetic acid, for example, to achieve an effective kill at lower concentrations of traditional germicides and this way minimise possible milk contamination with antimicrobial and/or, to maintain animal skin good condition by preventing skin irritation, which may occur when "aggressive' antimicrobial products are used. The composition may be formulated for topical application to the udder/teat region of a lactating animal. The composition may be formulated as a liquid or semi-liquid dip or spray.

The animal may be a non-human animal. The non-human animal may be lactating cow, goat or sheep.

The composition may act to sterilise the area to which the composition is applied by killing microbes on the surface of the udder/teat region or regions.

The treatment process may entail milking the animal, coating the udder/teat regions topically with the composition after milking, allowing the composition to dry and so forming a layer of persistent barrier film on the teats.

The composition may be applied topically by painting, foaming, creaming, dipping or spraying. Spraying may be via a pump action atomiser or a nebuliser or an aerosol.

The composition may also provide a barrier to microbes between the environment and udder/teat region by forming a film against the udder/teat. The film also prevents growth of microbes at or on the surface of the udder/teat.

The composition may be used for prophylactic treatment of a dairy animal's teats to provide a long lasting persistent protective germicidal barrier film that demonstrates persistence between milking. The film may be a continuous, uniform persistent barrier. The composition may have sufficient longevity to retain a sterilisation effective amount on the teat for a time period of at least 6 hours. The time period may be at least about 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15 hours. As may be appreciated, this time period equates to the time period between milking be that once or twice daily milking.

The composition may decrease the number of microbes at the treated site by at least a three-log reduction. The composition may decrease the number of microbes by at least a four-log reduction. The composition may decrease the number of microbes sufficient to substantially completely kill all microbes present, that is, a greater than a five-log (99.999%) reduction. The composition can however be tailored to provide lesser or greater log reduction as desired by varying the concentration of the key active agents.

The composition may be anti-microbial to pathogens.

In one embodiment, the composition may be tailored to sterilise the udder/teat to which the composition is applied against microbes associated with mastitis. In one embodiment, the mastitis microbes include: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Escherichia coli, Corynebacterium bovis and combinations thereof.

Despite the above, use of the composition is not limited to use against mastitis, and the composition may be used generally to treat or protect against any infectious skin condition and maintain skin condition.

The microbes may be bacteria. The microbes may be gram positive bacteria. The microbes may be gram negative bacteria.

The composition may be synergistically effective against C. bovis to a level comparable or even beyond that of iodine alone. As may be appreciated, this is unexpected as this particular microbe presents a particularly difficult challenge to sterilise, particularly to the extent of a 3-log reduction.

The inventor has identified that the above composition may be storage stable over time. The composition may be storage stable for longer than art teat spray compositions. It is anticipated that the composition will be storage stable for at least about 2 years when stored at ambient conditions. By contrast, art compositions such as iodine-based compositions are only storage stable for approximately 1 year.

(a) 30-69% w/v of the total agent concentration dodecyl benzene sulphonic acid; and (b) 30-50% w/v of the total agent concentration lactic acid; and

(c) 1 -20% w/v of the total agent concentration sodium citrate; and

In a third aspect, there is provided a method of preventing and/or treating a microbial infection in a non-human animal by topical administration of a composition substantially as described above. In a fourth aspect, there is provided a method of sterilising the udder region or regions of a lactating non-human animal by the step of topically applying the composition substantially as described above to the udder region or regions of the animal.

(a) at least one sulphonic acid; and

(b) lactic acid; and

(c) at least one citrate compound.

In a sixth aspect, there is provided a method of sterilising a skin area from C. bovis infection by topical application to the skin area of a non-human animal, a pharmaceutically and

(a) at least one sulphonic acid; and

(b) lactic acid; and

(d) at least one citrate compound.

A method of improving milk quality in milk derived from a lactating non-human animal by the step of:

(a) applying to the teat area of the non-human animal a liquid composition including a therapeutically effective amount of: at least one sulphonic acid; and lactic acid; and at least one citrate compound.

In the methods above, the applied region may be allowed to dry post application during which time period, a film forms on the treated region(s).

Methods of topical application may include: painting, dipping, foaming, creaming and spraying. Spraying may be via a pump action atomiser or a nebuliser or an aerosol.

Advantages of the above composition, method of manufacture, method of treatment and use are varied including the unexpected synergy in killing micro-organisms and the unexpected synergy in reducing the amount of other antimicrobial agents used (if used at all). The composition uses known food safe compounds that are benign and not noxious to humans or animals. In addition the compounds do not leave a taste in milk should contamination occur. The compounds used are inexpensive and have dual active and ancillary effects such as avoiding skin irritation and potential opportunistic infections that may result from irritation.

The composition also may assist with wound healing by either treating the infection directly, assisting in the treatment process e.g. by co-treatment with antibiotics and/or prevention of further infection.

The embodiments described above may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the embodiments relates, such known equivalents are deemed to be incorporated herein as of individually set forth,

Where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

WORKING EXAMPLES

The above described compositions, methods and use are now described with reference to examples illustrating the anti-microbial composition.

EXAMPLE 1

The synergistic effect between the above described three agents was verified by comparison with formulations where the active ingredient consisted of only two acids or one acid and salt in different combinations of dodecyl benzene sulphonic acid, lactic acid and sodium citrate. Formulations were prepared as follows:

Formulation 1 - Dodecyl benzene sulphonic acid, lactic acid;

Formulation 2 - Dodecyl benzene sulphonic acid, sodium citrate;

Formulation 3 - Lactic acid, sodium citrate;

Formulation 4 - Dodecyl benzene sulphonic acid, lactic acid, sodium citrate.

All formulations contained same amount of emollients and other additives.

Method

The formulations were tested on their performance towards Staphylococcus aureus,

Streptococcus agalactiae, Streptococcus uberis, Escherichia coli, and Corynebacterium bovis. These bacteria were chosen as they are common factors in mastitis being a major infection problem in lactating animals. C. bow^'s in particular presents a particularly strong challenge as only the strongest of antimicrobial agents is effective against this bacterium but often with their own drawbacks.

AOAC Official Method 960.09 was used to test the biocidal effects of the various formulations, AOAC as published by the Association of Analytical Chemists (AOAC International) in 2000 (Association of Official Analytical Chemists. 1990 (Official Methods of Analysis, Pages 138-140 in Germicidal and Detergent Sanitizing Action of Disinfectants 960.09, Vol. I. 15th ed. AOAC, Arlington, Va.). The AOAC Official Method 960.09 offers the advantages of being highly reproducible and because this method is a well established way to evaluate disinfecting and antimicrobial performance in the presence of excess liquid.

According to the test methodology of AOAC 960.09, AOAC-specified cultures of gram-positive and gram-negative bacterium are grown in appropriate broth, and then concentrated to approximately 1 x 10¹⁰ cfu/ml. In duplicate, for each microorganism, 1 ml of the concentrated culture is inoculated into an Erienmeyer flask containing 99 ml of the test product, for the specified contact time. Separately, 1 ml of the concentrated culture is inoculated into an Erienmeyer flask containing 99 ml of non-germicidal buffer, to determine the initial concentration of microorganisms. After the contact time has elapsed, aliquots of the microbe/product mixture are removed from the flask and analysed for surviving microorganisms by dilution and plating, coupled with an appropriate chemical neutralization strategy. Agar plates are incubated for a sufficient period. After incubation, the concentration of surviving microorganisms in the test flasks is compared to that in the control (initial concentration) flasks.

Results

Historically, combinations of separate organic acids and surfactants in topical solutions evidenced little antimicrobial efficacy. In some of these instances, the additives interfered with the efficacy. Thus, even when successful kills might be obtained using only lactic acid along with a surfactant in an aqueous solution, that effectiveness would diminish when the application required additives (e.g. viscosity control and opacifying agents, barrier forming agents, etc.). This interference made these combinations unsuitable for real-world applications, especially ones in which some contact with animal skin is intended (e.g. teat-dip/spray topical germicides).

It is also well known fact that compositions based on alkylbenzene sulphonic acids do not show antimicrobial activity towards Corynebacterium bovis.

Through experimentation, it was surprisingly discovered that the combination of dodecyl benzene sulphonic acid, lactic acid and citric acid in citrate form provide a synergistic result enabling greater than three-log reductions (99.9%) towards all tested bacteria, including

Corynebacterium bovis. The formulation also avoids conventional oxidisers which are more harmful to the animals' skin and has superior antimicrobial properties than other formulations (Formulation 1 , 2, 3).

For example, Formulation 3 gave only a 1.6-log reduction when tested against Staphylococcus aureus and a 1.4-log reduction when tested against Streptococcus agalactiae. Formulations 1 and 2 gave a comparable log reduction as Formulation 4 except when treating the more persistent microbe Corynebacterium bovis where none of the trial formulations showed any major sterilisation effect apart from Formulation 4 which exhibited a synergistically high sterilisation effect.

Full results are shown below in Table 1.

Table 1 - Comparison Experiments

EXAMPLE 2

Shown below are the results of experiments carried out to determine the efficacy of proposed antimicrobial composition against two contagious mastitis causing bacteria : Staphylococcus aureus and Streptococcus agalatiae, two environmental mastitis causing bacteria: Escherichia coli and Streptococcus uberis, and also Corynebacterium bovis. The antimicrobial agents in the compositions disclosed below in Table 2 comprise combinations of dodecyl benzene sulphonic acid, lactic and citric acid sodium salt along with numerous additives that may be included in a product for topical use.

One advantage attributable to dodecyl benzene sulphonic acid that is unrelated to efficacy is that it is entirely biodegradable and thus environmentally friendly. So much so, that it meets the European Surfactant Detergent Regulation requirements. This makes its inclusion in antimicrobial compositions more readily acceptable than other potential ingredients.

Results of the trials can be seen in Table 2 below. Table 2 - Efficacy of Formulation 4

As can be seen from Table 2, the proposed formulation surprisingly shows high antimicrobial activity even against Corynebacterium bovis, unlike other compositions based on linear benzene sulfonic acid (NMC Newsletter "Udder Topics", December 2000, National Mastitis Council 1999).

EXAMPLE 3

To investigate the proposed formulation ability to enhance other traditional antimicrobial compositions properties, a formulation was prepared consisting of an iodine based antimicrobial composition at a ¼ of recommended rate and half of recommended rate of the Formulation 4 composition. Efficacy of prepared ready to use solution was checked towards Escherichia coli, Staphylococcus aureus, and Streptococcus uberis. Results of the trial can be seen in Table 3 below. Table 3- Enhanced Effects With Traditional Antimicrobials

The results above show that even at half of recommended application rate the Formulation 4 composition significantly increases traditional and most of the time expensive antimicrobial compositions properties and allows to use them at half or even lower rate than recommended by their manufacturer. While this has a significant economical effect, it decreases if not eliminates animal skin irritation, and provides better skin conditioning for a much longer period of time. EXAMPLE 4

An example of the proposed antimicrobial composition is described. The composition includes:

Dodecyl benzene sulphonic acid - 105 g/L (calculated on 100% active basis)

Lactic acid 88% solution- 08 g/L

Sodium citrate - 1 1 .145 g/L

Glycerine - 150 g/L

PVP K30- 2 g/L

Lanolin ethoxylated - 10 g/L

Alkyl alcohol ethoxylated - 12 g/L

Sodium hydroxide - to pH 3.0-4.0

Water - to balance

EXAMPLE 5

An example_o.fthe_pr.opQsed_antiroi_.crobiai composition is_. described. The composition includes: Dodecyl benzene sulphonic acid 96% -103.125 g/L

Lactic acid 88% - 96.6 g/L

Sodium citrate - 13.37 g/L

Sorbitol - 80 g/L

Monopropylene glycol - 160 g/L

Alkyl polyglucoside ( Agnique PG8107) - 8 g/L

Pluronic PE 6200 - 10 g/L

Polyvinyl alcohol - 3 g/L

Water - to balance

EXAMPLE 6

The composition was further tested to determine the efficacy against mastitis in dairy cows. Somatic cell count (SCC) in dairy milk from was used as a measure of effectiveness of the composition. Somatic cell count is an indicator of milk quality and the SCC level increases in response to pathogenic bacteria, particularly those that cause mastitis. Higher SCC levels correspond to poorer quality milk.

Method

Field trials were completed in the Waikato and Southland regions of New Zealand representing two very different climates and different water usages. A sample of 800 dairy cows was selected for trial.

Cows were initially treated with the control treatment i.e. iodine or chlorhexidine generic teat sprays and then sprayed over a time period of 10 days using a formulation containing the three acids as described in Example 4 above. After the 10-day trial period elapsed, the original treatment regime using iodine or chlorhexidine generic teat sprays was followed again. Teat treatment occurred after milking.

During the pre-trial time period, the trial period itself and the post trial time period, somatic cell count (SCC) was monitored in milk produced from the cows.

The dose rate of iodine based teat sprays used was 2 to 2.3 g/l active iodine per dose and for chlorhexidine sprays, the dose rate was 4.0 to 5.0 g/l or a dilution rate of 1 :4 to 1 :9 with water depending on the formulation. The dose rate of the composition described herein was 1 :4 dilution providing 24.3g/l dodecyl benzene sulphonic acid and the other acids in proportion with this dose. Results

Prior to the trial period commencing the SCC levels were 358,000 cells per ml. While the trial was in progress, the SCC level dropped to 310,000-314,000. After the trial was complete, the SCC again increased to 361 ,000. Expressed in terms of percentages, the drop in SCC level of 42,000 - 56,000 cells per ml during the trial time period equates to an 11 to 16% drop.

The findings showed a measurable an important improvement in milk quality as measured by a drop in somatic cell count during the trial period. The results found were an improvement over existing art teat sprays therefore demonstrating the composition described herein gave improved anti-microbial properties over existing art formulations.

The mechanism of action is thought to be different to existing art formulations possibly due to better penetration into the skin and canals and as the composition described has better biocompatibility and confers a synergistic anti-microbial effect, particularly against microbes not well managed by art formulations.

Given the importance of somatic cell count and its connection with milk quality and hence milk value, the composition described herein may have considerable benefits commercially over existing art treatments as it demonstrates highly effective anti-microbial activities and it is biodegradable.

Aspects of the compositions and methods have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the claims herein.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutically and physiologically acceptable anti-microbial composition including a therapeutically effective amount of the active agents:

(a) at least one sulphonic acid; and

(b) lactic acid; and

(c) at least one citrate compound;

2. The composition as claimed in claim 1 wherein the active agents comprise 10-50% w/v of the total composition.

3. The composition as claimed in claim 1 or claim 2 wherein the sulphonic acid is dodecyl benzene sulphonic acid included at a rate of 30-69% w/v of the total active agent concentration.

4. The composition as claimed in any one of the above claims wherein the lactic acid is included at a rate of 30-50% w/v of the total active agent concentration.

5. The composition as claimed in any one of the above claims wherein the citrate salt is sodium citrate included at a rate of 1-20% w/v of the total active agent concentration.

6. The composition as claimed in any one of the above claims wherein the composition forms a sterilisation effective film on the area to which the composition is applied for at least 6 hours.

7. The composition as claimed in any one of the above claims wherein the composition is biodegradable.

8. The composition as claimed in any one of the above claims wherein the composition has a synergistic anti-microbial activity against C. bovis bacteria.

9. The composition as claimed in any one of the above claims wherein the active agents claimed act to synergistically reduce the need for other anti-microbial agents.

10. The composition as claimed in any one of the above claims wherein the composition is formulated as a spray for application to the teat or teats of a lactating non-human animal.

11. A method of treating a microbial infection in a non-human animal by topical administration of a composition as claimed in any one of the above claims.

12. A method of sterilising the skin of a non-human animal by topical administration to the non-human animal of a pharmaceutically and physiologically acceptable liquid composition including a therapeutically effective amount of the active agents:

(a) at least one sulphonic acid; and

(b) lactic acid; and

(c) at least one citrate compound.

13. The method as claimed in claim 12 wherein the active agents comprise 10-50% w/v of the total composition.

14. The method as claimed in claim 12 or claim 13 wherein the sulphonic acid is dodecyl benzene sulphonic acid included at a rate of 30-69% w/v of the total active agent concentration.

15. The method as claimed in any one of claims 12 to 14 wherein the lactic acid is included at a rate of 30-50% w/v of the total active agent concentration.

16. The method as claimed in any one of claims 12 to 15 wherein the citrate compound is sodium citrate included at a rate of 1-20% w/v of the total active agent concentration.

17. The method as claimed in any one of claims 12 to 16 wherein the composition forms a sterilisation effective film on the area to which the composition is applied for at least 6 hours.

18. The method as claimed in any one of claims 12 to 17 wherein the composition is biodegradable.

19. The method as claimed in any one of claims 12 to 18 wherein the composition has a synergistic anti-microbial activity against C. bovis bacteria.

20. The method as claimed in any one of claims 12 to 19 wherein the active agents act to synergistically reduce the need for other anti-microbial agents.

21. The method as claimed in any one of claims 12 to 20 wherein the composition is formulated as a spray for application to the teat or teats of a lactating non-human animal.

22. A method of sterilising a skin area from C. bovis infection by topical application to the skin area of a non-human animal, a pharmaceutically and physiologically acceptable liquid composition including a therapeutically effective amount of the active agents:

(a) at least one sulphonic acid; and

(b) lactic acid; and

(c) at least one citrate^ompound.

23. A pharmaceutically and physiologically acceptable anti-microbial composition including 10-50% w/v of the total composition active agents, wherein the active agents include:

(b) 30-50% w/v of the total agent concentration lactic acid; and

(c) 1-20% w/v of the total agent concentration sodium citrate; and

wherein the composition is a liquid formulated for topical application to a non-animal.

24. A method of improving milk quality in milk derived from a lactating non-human animal by the step of:

25. Use of a composition as claimed in any one of claims 1 to 10 in the manufacture of a medicament for the treatment of a microbial infection in an animal.