WO2008031105A1 - Polymeric guanidine salt-based germicides - Google Patents

Abstract

Disclosed is an antimicrobial liquid composition that contains at least one, or a mixture of Poly (HexaMethylenediamine) Guanidinium hydrochloride (PHMG) and Poly [2- (2- Ethoxy) -Ethyl] Guanidinium hydrochloride (PEEG). In some embodiments; the composition is applied as part of a teat-dip application. In yet further embodiments, the activity of the composition is enhanced by maintaining pH levels higher than 8. In other embodiments, a surfactant is included.

Description

POLYMERIC GUANIDINE SALT-BASED GERMICIDES

RELATED APPLICATIONS

[0001] This application claims the benefit of priority to commonly-owned and copending U.S. Provisional Patent Application Nos. 60/843,113, filed 8 September 2006, 60/888,243, filed 5 February 2007, and 60/970,800, filed 7 September 2007, each of which is incorporated herein by reference.

BACKGROUND

1. Field of the Invention

[0002] The present invention pertains to biocide compositions of the type that may be used to control or destroy pathogenic microorganisms. More particularly, various antimicrobial agents are shown to work with cooperative effects against microorganisms in a wide variety of applications.

2. Description of the Related Art

[0003] Antimicrobial compositions are used to reduce the risk 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. They may be used on human skin to reduce the transmission of disease or infection, as surgical scrub solutions, or hand sanitizers. The compositions may also be used in veterinary applications for the control or prevention of hoof diseases and mastitis. Prevention of mastitis is also a major goal of the dairy industry. Contact of the bovine or ovine mammary gland with pathogenic microorganisms, usually bacteria but occasionally yeast or fungi, can result in the disease of mastitis.

[0004] Mastitis is the single most costly disease affecting the dairy industry. Annual economic losses due to mastitis approximate $185 per dairy animal. This totals to approximately $1.7 billion annually for the entire United States market. 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. [0005] To reduce mastitis, commercial teat dips 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. Teat dips applied subsequent to milking may form a thick composition, film or barrier that remains on the teat until the next milking, which is generally 8 to 12 hours later.

[0006] Commercially available teat dips may be divided into two primary classifications, namely, non-barrier and barrier dips. The non-barrier teat dips are strictly antimicrobial and are applied to kill microorganisms on the surface of the teat skin. By design, the microbiological effect is substantially immediate, targeting the contagious organisms that may be transferred between animals during the pre- milking, milking and post milking process. The barrier dips may also be antimicrobial and are applied to form a prophylactic film or coating that may prevent microbes from contacting the teat.

[0007] Teat dips have used a variety of antimicrobial agents. United States Patent No. 2,739,922 issued to Shelanski describes the use of polymeric N- vinyl pyrrolidone in combination with iodophors. United States Patent No. 3,993,777 issued to Caughman et al. describes the use of halogenated quaternary ammonium compounds. United States Patent No. 4,199,602 issued to Lentsch describes the use of iodophors, chlorine releasing compounds (e.g. alkali hypochlorite), oxidizing compounds (e.g. hydrogen peroxide, peracids), protonated carboxylic acids (e.g. heptanoic, octanoic, nonanoic, decanoic, undecanoic acids), and nitroalkanols. United States Patent No. 4,434,181 issued to Marks, Sr. et al. describes the use of acid anionics (e.g. alkylaryl sulfonic acids), chlorine dioxide (from alkali chlorite), and bisbiguanides such as chlorhexidine.

[0008] Some of the available teat dip 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. Additionally, the use of overly powerful disinfectants may contribute to the mastitis problem by causing irritation of the teat skin, thus providing an opportunistic site which promotes infection. The Lentsch '602 patent recognizes that iodophors and such chlorine-based biocides as hypochlorite, chlorine dioxide, and hypochlorous acid have achieved the widest commercial acceptance; however, teat dips of the future may have to be iodine- free. Furthermore, the iodine-based and chlorine-based compositions may induce sensitized reactions in cow teats. Other biocides including chlorhexidine have become the focus of regulatory concern. On the other hand, less powerful teat dip agents, such as fatty acids and anionic surfactants, are often not broad enough in their antibacterial spectrum to provide complete germicidal protection.

[0009] From a consumption point of view, it is 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 which 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 and it is recommended that some populations, such as pregnant women, limit their intake. 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.

SUMMARY

[0010] In embodiments, the invention is an antimicrobial composition comprising a polymeric-guanidine salt-based antimicrobial agent. In one embodiment the composition is adapted for topical applications on an animal. In another embodiment, the formulation is used as part of a teat-dip process. Those skilled in the art will appreciate additional objects and advantages in the detailed description below. All references specifically disclosed in this specification are hereby incorporated by reference.

DETAILED DESCRIPTION

[0011] There will now be shown and described as a particular embodiment, an antimicrobial liquid composition that contains a cationic guanidine agent. In another embodiment, the agent includes at least one polymeric cationic guanidine salt. In yet another embodiment, the positively charged polymer includes at least one of Poly(HexaMethylenediamine) Guanidinium hydrochloride (PHMG) and Poly[2-(2-Ethoxy)-Ethyl] Guanidinium hydrochloride (PEEG). In other embodiments, the agent includes both PHMG and PEEG as a mixture. PHMG and PEEG mixtures, in some embodiments, can be in weight ratios from of 1 : 100 PHMG to PEEG to 100:1 PHMG to PEEG. In other embodiments, the ratios can be from 1 :20 PHMG versus PEEG to 20: 1 PHMG to PEEG. In still other embodiments, the PHMG versus PEEG weight ratios could be from 1 : 10 to 10: 1. In one embodiment, the biocides are a mixture of about a 3:1 ratio of PHMG salts and PEEG salts, and the mixture is readily commercially available under the trade name Akacid Plus^®. Akacid Plus^® is manufactured and commercially available from P. O. C. Oil Industry Technology Beratungsges m.b.H. in Vienna, Austria. General support for biocide polymers based on guanidine salts can be found in U.S. Patent No. 7,001,606.

[0012] In one embodiment, the antimicrobial liquid composition is applied topically. In other embodiments, the composition is applied as part of a teat-dip application. In yet further embodiments, the activity of the compositions is enhanced by maintaining pH levels higher than 8. In other embodiments, the compositions are used along with a surfactant. In still further embodiments, the compositions are used with a surfactant, and the pH level is enhanced to be between 8 and 10. In still further embodiments, the compositions are used with pH higher than 10 in hard surface applications.

[0013] The Akacid Plus^® biocide product is advantageous in that it is supported by European Biocidal Products Directive (BPD). Further, it has a broad antimicrobial spectrum covering hundreds of important strains of pathogens, fungi, and possibly viruses.

[0014] The germicidal action of the product is swift as the positively charged polymer surrounds and engages the negatively charged cell walls of the microorganism. This leads to the neutralization and reversal of the negatively charged walls, thus damaging the cytoplasmic membrane of the bacteria cell, as can be documented by an efflux of potassium ions and finally by the diffusion of nucleotides. No resistant strains to the biocide have been detected to date.

[0015] The product is particularly useful because it is non-toxic for humans, animals, and plants at moderate doses. Additionally, it is neutralized in anionic surroundings or soil, and thus, is biodegradable. Further, it is not irritating to the body surfaces, mucosa, or the eyes. Additionally, the product is easy to handle and store, and can be used in nebulizers. [0016] In one embodiment, the antimicrobial is included in a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may, for example, be water. In other embodiments, the antimicrobial may be included in a carrier-reduced or carrier-free concentrate.

[0017] The carrier may include one or more additives selected from a buffering agent, an emollient, a humectant, a preservative, a barrier forming agent, a surfactant or wetting agent, a viscosity control agent, a colorant, an opacifying agent, and any combinations thereof.

[0018] The antimicrobial compositions provide a substantial reduction in Gram positive and Gram negative bacteria, as well other numerous classes of microbes. For particular embodiments, the reduction may be on the order of a three or four log reduction or a substantially complete kill that is greater than a five log (99.999%) reduction. In other embodiments, the kill counts could be higher or lower.

[0019] A broader object of the disclosed instrumentalities is to provide biocide compositions that may be used, for example, according to any purpose for antibacterial or bactericidal properties. In a particular embodiment, the composition is intended to be used as a teat dip. In other embodiments the composition is intended to be used as a foaming agent, hand sanitizer, a skin cleanser, a surgical scrub, a wound care agent, a disinfectant, a mouthwash, bath/shower gel, a hard surface sanitizer and the like. In some embodiments, the compositions for skin applications have a pH of about 4 to about 12. In some embodiments, the composition could have a pH in the range of about 5 to 11. In still further embodiments, the compositions are adapted to have pH's in the range of about 6 to 10. Different uses, however, may make pH targets stray significantly from these ranges in other embodiments.

[0020] Methods of preparing compositions may involve dissolving a desired concentration of the antimicrobial and, optionally, any desired additives in a selected pharmaceutical carrier. The solution is then mixed, for example in a mixer, to form a final antimicrobial composition.

[0021] In embodiments, the composition is useable on a subject. As used herein, the term "subject" shall include humans and terrestrial animals. For example, the subject can be a domestic livestock species, a laboratory animal species, a zoo animal, a companion animal or a human. In a particular embodiment, "subject" refers more specifically to any lactating animal. In one embodiment, the subject is a cow.

[0022] The phrase "therapeutically effective amount" is intended to qualify the amount of the topical composition which will achieve the goal of decreased microbial concentration. "Therapeutically effective" may also refer to improvement in disorder severity or the frequency of incidence over no treatment.

[0023] The term "topical" shall refer to any composition which may be applied to the epidermis or other animal portion on which compositions might be applied. Topical shall also refer to compositions used as mouthwashes or adapted for other reachable internal portions.

[0024] The term "additive" shall mean any component that is not an antimicrobial agent or a pharmaceutical carrier. A pharmaceutical carrier is generally a bulk solvent used to dilute or solubilize the components of the composition. In embodiments, this carrier is water.

[0025] The terms "teat dip" or "teat dipping" shall be interpreted broadly and in accordance with the terminology used in the art of dairy farming. Thus, the composition is not only intended for dipping of the teats but it can, of course, be applied in other ways, such as by spraying or foaming and still fall within the recognized terms teat dip or teat dipping composition or agent.

[0026] As used herein unless other wise specified, the term "antimicrobial" describes a biocidal effect that may be, for example, an antibacterial, antifungal, antiviral, bacteriostatic, disinfecting, or sanitizing effect.

[0027] As shown in the examples below, the polymeric guanidine salt- based agent, e.g., Akacid Plus^®, can be used to make effective biocide compositions suitable for topical applications. This antimicrobial may be formulated using additional antimicrobial agents, barrier-forming agents, viscosity control agents, pH adjusting agents, wetting agents, opacifying agents, and carriers to make a wide variety of products.

[0028] It is anticipated that the antimicrobial above will be used with additional additive components. These antimicrobial ingredients may be formulated using additional antimicrobial agents, barrier-forming agents, foaming agents, viscosity control agents, pH adjusting agents, wetting agents, opacifying agents, chelating agents, skin conditioning agents and carriers to make a wide variety of products.

Additional Antimicrobial Agents

[0029] Traditional antimicrobial agents are the components of a composition that destroy microorganisms or prevent or inhibit their replication. In one aspect, the polymeric guanidine salt-based antimicrobial embodiments discussed above may be used to replace or eliminate the need for traditional antimicrobial agents in a wide variety of applications, In another aspect, antimicrobial compositions according to the disclosed embodiments below may be used in combination with these traditional antimicrobial agents, for example, to achieve an effective kill at lower concentrations of traditional antimicrobial agents.

[0030] Traditional antimicrobial agents include 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), acid anionics (e.g. alkylaryl sulfonic acids, aryl sulfonic acid, alkyl sulfonic acids, alkylaryl sulfuric acid, aryl sulfuric acid, alkyl sulfuric acid, alkylaryl sulfuric acid), 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, which is known commercially as Triclosan and may be purchased from Ciba Specialty Chemicals as IRGASAN™ and IRGASAN DP 300™. Another such antimicrobial agent is 4-chloro-3,5-dimethyl phenol, which is also known as PCMX and is commercially available as NIPACIDE PX and NIPACIDE PX-P. Other traditional germicides include formaldehyde releasing compounds such as glutaraldehyde and 2-bromo-2-nitro-l,3-propanediol (Bronopol), polyhexamethyl biguanide (CAS 32289-58-0), guanidine salts such as polyhexamethylene guanidine hydrochloride (CAS 57028-96-3), polyhexamethylene guanidine hydrophosphate (89697-78-9), and poly[2-(2-ethoxy)-ethoxyethyl]-guanidinium chloride (CAS 374572-91-5) and mixtures thereof.

[0031] In one embodiment, the disclosed germicides may be used in combination with traditional germicides such as copper sulfate, zinc sulfate, sulfamethazine, quaternary ammonium compounds, hydrogen peroxide and/or peracetic acid, for example, to achieve an effective kill at lower concentrations of traditional germicides.

Barrier Forming Agents

[0032] Barrier and film forming agents are those components of a teat dipping composition that 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 include thick creams or emollients (made with viscosity control agents), films, polymers, latex and the like. Some nonionic surfactants may help further enhance the barrier properties of a composition, in addition to contributing to surface wetting. Examples of such surfactants may include, without limitation, polyoxyethylene-polyoxypropylene glycol (marketed as Pluronic F 108). Another commonly used barrier agent is marketed as Pluronic P 105. A latex material that provides an effective covering of the teat is described in U.S. Pat. No. 4,113,854. Suitable barrier forming agents include, for example, latex, arabinoxylanes, glucomannanes, guar gum, johannistree gums, cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, carboxyethyl cellulose, carboxymethyl cellulose, starch, modified starch, hydrolyzed starch, hydroxyethyl starch, gum arabic, curdlan, pullulan, dextran, polysulfonic acid, polyacrylamide, high molecular weight polyacrylate, high molecular weight cross-linked polyacrylate, carbomer, glycerol, sodium alginate, sodium alginate cross-linked with calcium salt, xanthan gum, poly( vinyl alcohol) (PVA) and poly(N-vinylpyrrolidone) (PVP). Preferred embodiments for barrier- forming agents 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).

Foaming Agents

[0033] A foaming agent may be used in the disclosed antimicrobial compositions. A foaming agent aerates a liquid composition to produce foam that may increase surface area of the composition and improve contact with the surface to be treated (e.g., an animal hoof). Typically, a foaming agent is in the form of a compressed gas, or a material that will decompose to release gas under certain conditions. Suitable gases include but are not limited to nitrogen, argon, air, carbon dioxide, helium and mixtures thereof. In addition, solid carbon dioxide (dry ice), liquid nitrogen, hydrogen peroxide and other substances that release gas via a change in state or through decomposition are contemplated for use with the present compositions.

[0034] Typically, a high foaming surfactant such as sodium lauryl sulfate, dodecylbenzene sulfonic acid, sodium alkylaryl polyether sulfate, sodium lauryl ether sulfate, sodium decyl sulfate, cocamine oxide, Ci₂ - Ci₄ whole coconut amido betaines can be used to generate a stable foam. The foam is produced when agitation in the form of a compressed gas is mixed with the solution either by bubbling the gas into the solution or spraying the solution or solution-gas mixture through spray equipment. Suitable gases include but are not limited to nitrogen, air, carbon dioxide and mixtures thereof. Foam can also be generated by the mechanical action of animals walking through the composition, or by other mechanical means that mix atmospheric air with the composition. The composition can be applied by having animals walk through an area containing the foam or by having the animal walk through a footbath solution that has foam floating on top of the solution.

[0035] Surfactants are well known for foaming and are widely used as foaming agents in hand soap and manual/hand dishwashing detergents and such surfactants can be used as foaming agents in applications where foaming can boosts the performance and increase contact time of the composition to a particular substrates. Examples of such Suitable anionic surfactants can be chosen from a linear alkyl benzene sulfonic acid, a linear alkyl benzene sulfonate, an alkyl α-sulfomethyl ester, an α-olefm sulfonate, an alcohol ether sulfate, an alkyl sulfate, an alkylsulfo succinate, a dialkylsulfo succinate, and alkali metal, alkaline earth metal, amine and ammonium salts thereof. Specific examples are linear Ci_O-Ci₆ alkyl benzene sulfonic acid, linear Ci₀-Ci₆ alkyl benzene sulfonate or alkali metal, alkaline earth metal, amine and ammonium salt thereof e.g. sodium dodecylbenzene sulfonate, sodium Ci₄- Ci₆ α-olefm sulfonate, sodium methyl α-sulfomethyl ester and disodium methyl α- sulfo fatty acid salt. Suitable nonionic surfactants can be chosen from an alkyl polyglucoside, an alkyl ethoxylated alcohol, an alkyl propoxylated alcohol, an ethoxylatedpropoxylated alcohol, sorbitan, sorbitan ester, an alkanol amide. Specific examples include Cg-Ci₆ alkyl polyglucoside with a degree of polymerization ranging from 1 to 3 e.g., C₈-Ci_O alkyl polyglucoside with a degree of polymerization of 1.5 (Glucopon^® 200), C₈-Ci₆ alkyl polyglucoside with a degree of polymerization of 1.45 (Glucopon^® 425), Ci₂-Ci₆ alkyl polyglucoside with a degree of polymerization of 1.6 (Glucopon^® 625). Amphoteric surfactants can be chosen from alkyl betaines and alkyl amphoacetates. Suitable betaines include cocoamidopropyl betaine, and suitable amphoacetates include sodium cocoamphoacetate, sodium lauroamphoacetate and sodium cocoamphodiacetate. Alkyl amine oxides based on C 12-Cl 4 alkyl chain length feedstock such as those derived from coconut oil, palm kernel oil are also suitable foaming agents.

Viscosity Control Agents

[0036] Viscosity control agents may be added to formulate the antimicrobial compositions according to an intended environment of use. In one example, it is advantageous for some compositions to have an optimized 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, minimizes dripping of the product to avoid wastage and is particularly advantageous in teat dip compositions. Teat dip compositions may benefit from a preferred dynamic viscosity ranging from 1 cPs to 3000 cPs. Other applications including hard surface disinfectants have a preferred dynamic viscosity ranging from about 1 cPs to 300 cPs. In another example, the amount of viscosity control agents may be substantially reduced or even eliminated in other compositions, such as surface or floor disinfectants where easy cleanup is desired. An intermediate or medium viscosity composition may be useful in a hand cleaner or personal care product. It is seen from these examples that the antimicrobial compositions may be formulated for a wide variety of applications by altering the amount of viscosity control agents. The viscosity referred to throughout this application is Brookfield viscosity measured in cPs by a Brookfield LV viscometer at ambient temperature (25⁰C) with a spindle # 2 @ 3 to 30 rpm. In various embodiments, a thickener may be added to achieve a viscosity range of from 50 cPs to 10000 cPs, or from 100 cPs to 4000 cPs.

[0037] Suitable viscosity control agents include 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 sulfate, whey, gelatin, chitosan, chitosan derivatives, polysulfonic acids and their salts, polyacrylamide, and glycerol. Preferred viscosity controlling agents are, different types of cellulose and derivatives thereof, particularly hydroxyalkyl cellulose, methyl cellulose, and glycerol. High molecular weight (MW > 1,000,000) cross-linked polyacrylic acid type thickening agents are the products sold by B. F. Goodrich (now Lubrizol) under their Carbopol^® trademark, especially Carbopol 941, which is the most ion-insensitive of this class of polymers, and Carbopol^® 940 and Carbopol^®934. The Carbopol^® resins, also known as "Carbomer", are reported in U.S. Pat. No. 5,225,096, and are hydrophilic high molecular weight, cross-linked acrylic acid polymers. Carbopol^® 941 has a molecular weight of about 1,250,000, Carbopol^® 940 has a molecular weight of approximately 4,000,000, and Carbopol 934 has a molecular weight of approximately 3,000,000. The Carbopol^® resins are cross-linked with polyalkenyl polyether, e.g. about 1 % of a polyallyl ether of sucrose having an average of about 5.8 allyl groups for each molecule of sucrose. Further detailed information on the Carbopol^® resins is available from B.F. Goodrich (Lubrizol), see for example, the B. F. Goodrich catalog GC-67, Carbopol^® Water Soluble Resins. Clays and modified clays such as bentonite or laponite can also be used as thickeners. Co-thickeners are often added to improve the stability of the gel matrix, for example, colloidal alumina or silica, fatty acids or their salts may improve gel stability. Typical viscosity control ingredients include xanthan gum, carboxymethyl cellulose, sodium alginate, sodium alginate cross-linked with calcium salt, polysulfonic acids and their salts, polyacrylamide, polyvinyl alcohol (PVA), hydroxyethyl cellulose and polyN-vinylpyrrolidone (PVP).

pH Adjusting Agents

[0038] A composition pH value may be selectively adjusted by the addition of acidic or basic ingredients. Generally, an acidic pH is preferred. Suitable acids for use as pH adjusting agents may include, for example, lactic acid, citric acid, phosphoric acid, phosphorous acid, sulfamic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid and hydrochloric acid. Mineral acids may be used to drastically lower the pH. The pH may be raised or made more alkaline by addition of an alkaline agent such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate or combinations thereof. Traditional acid buffering agents such as citric acid, lactic acid, phosphoric acid may also be used to maintain a desired pH, e.g., within the desired ranges discussed above.

Wetting Agents

[0039] Wetting agent(s) or surface active agent(s) are also known as surfactants. Typical wetting agents are used to wet the surface of application, reduce surface tension of the surface of application so that the product can penetrate easily on the surface and remove unwanted soil. 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.

[0040] Suitably effective surfactants used for wetting may include anionic, cationic, nonionic, zwitterionic and amphoteric surfactants. Wetting agents and surfactants used in the inventive applications can be high foaming, low foaming and non foaming type. Suitable anionic surfactants can be chosen from a linear alkyl benzene sulfonic acid, a linear alkyl benzene sulfonate, an alkyl α-sulfomethyl ester, an α-olefin sulfonate, an alcohol ether sulfate, an alkyl sulfate, an alkylsulfo succinate, a dialkylsulfo succinate, and alkali metal, alkaline earth metal, amine and ammonium salts thereof. Specific examples are linear Ci₀-Ci₆ alkyl benzene sulfonic acid, linear CiQ-Ci₆ alkyl benzene sulfonate or alkali metal, alkaline earth metal, amine and ammonium salt thereof e.g. sodium dodecylbenzene sulfonate, sodium Q₄- C] ₆ α-olefin sulfonate, sodium methyl α-sulfomethyl ester and disodium methyl α- sulfo fatty acid salt. Suitable nonionic surfactants can be chosen from an alkyl polyglucoside, an alkyl ethoxylated alcohol, an alkyl propoxylated alcohol, an ethoxylatedpropoxylated alcohol, sorbitan, sorbitan ester, an alkanol amide. Specific examples include C₈-Ci₆ alkyl polyglucoside with a degree of polymerization ranging from 1 to 3 e.g., C₈-Ci_O alkyl polyglucoside with a degree of polymerization of 1.5 (Glucopon^® 200), C₈-Ci₆ alkyl polyglucoside with a degree of polymerization of 1.45 (Glucopon 425), Ci₂-Cj₆ alkyl polyglucoside with a degree of polymerization of 1.6 (Glucopon 625), and polyethoxylated polyoxypropylene block copolymers (poloxamers) including by way of example the Pluronic^® poloxamers commercialized by BASF Chemical Co. Amphoteric surfactants can be chosen from alkyl betaines and alkyl amphoacetates. Suitable betaines include cocoamidopropyl betaine, and suitable amphoacetates include sodium cocoamphoacetate, sodium lauroamphoacetate and sodium cocoamphodiacetate.

Opacifying Agents and Dyes

[0041] An opacifying agent or dye is optionally included in the present compositions. For example, color on a teat tells a farmer that a particular cow has been treated. To preclude any problems with possible contamination of milk, it is preferred that only FD&C Certified (food grade) dyes be used. There are many FD&C dyes available which are FD&C Red #40, FD&C Yellow #6, FD&C Yellow #5, FD&C Green #3 and FD&C Blue #1. Dyes used either alone or in combination are preferred. D&C Orange #4 can also be used. Titanium dioxide (TiO₂) is widely used as an opacifier and can also be used in combination with various colorants.

Preservatives and Chelating Agents

[0042] Some known teat dips and hand sanitizers include ethylenediaminetetraacetic acid (EDTA) and/or its alkali salts which can act as a chelating agent to remove metal ions from hard water. The metal ions, if not removed from the composition, serve as reaction sites for enzymes within the bacteria; the metalloenzyme reactions produce energy for bacterial cell replication. Other traditional preservatives are widely used, for example, paraban, methyl paraban, ethyl paraban, glutaraldehyde, etc. Preservatives such as an alcohol can also be added. The alcohol, in embodiments, may be benzyl alcohol.

Skin Conditioning Agents

[0043] Skin conditioning agents may also be optionally used in the disclosed compositions. Skin conditioning agents may provide extra protection for human or animal skin prior to or subsequent to being exposed to adverse conditions. For example, skin conditioning agents may include moisturizers, such as glycerin, sorbitol, propylene glycol, D-Panthenol, Poly Ethylene Glycol (PEG) 200-10,000, Poly Ethylene Glycol Esters, Acyl Lactylates, Polyquaternium-7, Glycerol Cocoate/Laurate, PEG-7 Glycerol Cocoate, Stearic Acid, Hydrolyzed Silk Peptide, Silk Protein, Aloe Vera Gel, Guar Hydroxypropyltrimonium Chloride, Alkyl Poly Glucoside/Glyceryl Luarate, shea butter and coco butter; sunscreen agents, such as titanium dioxide, zinc oxide, octyl methoxycinnamate (OMC), 4-methylbenzylidene camphor (4-MBC), oxybenzone and homosalate; and itch-relief or numbing agents, such as aloe vera, calamine, mint, menthol, camphor, antihistamines, corticosteroids, benzocaine and paroxamine HCl.

Pharmaceutical Carriers

[0044] A typical carrier or matrix for an antimicrobial composition is deionized water, although one skilled in the art will readily understand that other solvents or compatible materials other than water may be used to achieve the effective concentrations of germicidal agents. In some embodiments, a composition may contain at least about 60% water and preferably at least about 70% water by weight based on the total weight of the composition. Propylene glycol, glycol ethers and/or alcohols can also be used as a carrier either alone or in combination with water.

Materials and Reagents

[0045] The test bacteria were Escherichia coli (ATCC 11229), which were originally isolated from mastitis infection and obtained on commercial order from Mastitlaboratoriet, SVA, and Staphylococcus aureus (ATCC6538 from Microbiologies, St. Cloud, MN), which was also isolated from mastitis infection.

Testing of Antimicrobial Activity [0046] With respect to Tables I- VI below, various standardized test methods were used for comparatively testing the kill efficacy of antimicrobial agents. The preferred standard is defined as AOAC Official Method 960.09, as published by the Association of Analytical Chemists (AOAC International) in 2000. Europeans tend to use other standards for this same purpose, such as the EN 1040, EN 1656 and EN14885 test methods. All of these standards are incorporated by reference to the same extent as though fully disclosed herein.

[0047] According to a modified ENl 656 dilution neutralization method, freeze dried E. coli (ATCC 11229) and S. aureus (ATCC 6538) were hydrated, grown for four days and transferred. Then bacteria were diluted to form a suspension having an initial concentration of about 10 cfu/mL. Sterilized skimmed milk was used as an interfering substance in all testing. One mL of milk and 1 mL of bacterial suspension were mixed and left in contact for 2 minutes at 25 ⁰C. Eight mL of the solutions described below in Tables 1 and 2 were then added to the mixture and left in contact for 30 seconds at 25 ⁰C. One milliliter of the resulting solution was removed and neutralized, and then four successive dilutions were made. Samples from each dilution were plated in duplicate and agar was added. The initial bacterial suspension was diluted and plates were made in duplicate for controls. When the agar had solidified the plates were set at 37 ⁰C for 48 hours. The plates with bacterial populations between 25 and 250 were counted via visual inspection, and results were expressed as logarithmic reductions.

Irritation Testing

[0048] As will be seen below, Blood Cell Irritation tests were performed to determine if some particular representative compositions would be mild enough for topical applications. These tests involved separating red blood cells and then exposing them to the compositions. The tests used for the compositions addressed in Tables 1 -2 included two measurements made on cow's blood. In the tests, fresh calf blood samples were obtained; 50 mL of sodium citrate buffer was added to every 450 mL of blood and mixed. The blood was then centrifuged to isolate red blood cells (RBC), which were then washed with sodium citrate buffer, and centrifuged several times to remove white cells and plasma, according to a known method. The red blood cells were placed into containers for use in testing the disclosed antimicrobial compositions.

[0049] Red blood cells were treated with water, centrifuged and then, using a UV spectrophotometer, the absorption at 560 nm was measured in order to determine complete cell denaturation (Hi₀₀). The product to be tested was then diluted in the range of 5000 ppm to 60000 ppm, blood cells were added to these dilutions, centrifuged and the absorption at 560 nm was measured by UV spectrometry. Haemolysis Values (H₅₀) were determined by plotting absorption versus concentration. The H₅o value represents the product concentration (expressed in ppm) at which half of the blood cells are denatured.

[0050] Product Haemolysis Values (H₅₀); Product Denaturation Index Values (DI); and Lysis/Denaturation Ratios (L/D) were determined for the compositions using known methods. Descriptions of these methods were disclosed by Wolfgang J. W. Pape, Udo Hoppe: In vitro Methods for the Assessment of Primary Local Effects of Topically Applied Preparations, Skin Pharmacol. (1991), 4, 205-212, which is incorporated herein by reference. The haemolysis - or tendency of the red blood cells to rupture when in contact with the test product - was measured by the half-haemolysis value H₅₀. The denaturation of protein caused by the test product was measured by the denaturation index (DI). For DI measurements, a 1000 ppm solution of sodium lauryl sulfate solution was used as a reference. The overall irritation value for a product was determined by the ratio of the H₅₀/DI which is referred to as the lysis/denaturation quotient. The overall irritation score is given by the lysis/denaturation value which is calculated by the equation: L/D = H5₀ (measured in ppm) / DI (measured in %).

[0051] The H₅₀ score which measures haemolysis alone usually shows a similar irritation correlation to the L/D ratio. The higher the ppm value for H₅₀ the less irritating the product. A crude scale is H₅₀ > 500ppm (non-irritant); 120-500 (slight irritant), 30 - 120 (moderate irritant), 10 - 30 (irritant), 0-10 (strong irritant).

[0052] The DI score which measures denaturation of protein also shows a correlation to the L/D ratio. A crude scale is DI 0-5% (non- irritant); 5 - 10% (slight irritant), 10 - 75% (moderate irritant), 75 - 100% (irritant), and >100% (strong irritant). [0053] Although the H₅₀ and DI values may be of use in the interpretation of the results, the L/D ratio is the primary value used to determine irritation. This method is best suited to comparing two or more products and determining which product is likely to cause the least irritation to skin and eyes. In terms of indication, an L/D value greater than 100 is an indication that the composition is a non-irritant; levels between 10 and 100 are considered slight irritants; levels between 1 and 10 are considered moderate irritants; level between 0.1 to 1 are considered irritants; and levels lower than 0.1 are considered strong irritants.

Results

[0054] Through experimentation, it has been discovered that polymeric guanidine agents when used with certain additives in an aqueous solution are capable of in are capable of effectively killing bacteria (e.g., producing five-log reductions or higher) while avoiding skin irritation, and maintaining sufficient viscosity for use in a teat dip or other like applications.

[0055] Shown in Tables I- VI below are the results of experiments carried out to determine the efficacy of various antimicrobial compositions against E. coli and S. aureus. The formulations below all use a polymeric guanidine salt-based antimicrobial agent which includes Poly-(HexaMethylendiamineGuanidinium hydrochloride) (PHMG) and Poly- [2-(2-Ethoxy) Ethyl Guanidinium hydrochloride] in a mixture of about a 3:1 ratio of PHMG salts and PEEG salts, and the mixture is readily commercially available under the trade name Akacid Plus . Akacid Plus® is commercially available in a concentration of 25% (Akacid Plus 25%), meaning that only a quarter of the amounts listed in the Tables are actually active ingredient.

[0056] The embodiments in each of the tables show use of the Akacid Plus, along with numerous additives which design the product, in one embodiment, for topical uses, as well as a well-known ethoxylated nonionic surfactant sold commercially as Neodol 91-8, as it is reflected in the Tables. Here Neodol 91-8 is included as a potential barrier and/or film forming agent which, in one embodiment, adapts the formulation for teat-dip applications. Another component, sodium hydroxide (NaOH) is used here as a pH adjusting agent. The rest of the solution is water. TABLE I

B D E F H K

[0057] As can be seen from Table I, significant kill properties were observed at 30 seconds, and for the embodiments tested, good kill properties were observed at 1 and 5 minutes. Further formulation embodiments are shown below in Tables II-IV in which Akacid Plus amounts and pH levels are manipulated to provide further results.

TABLE II

B C D F G H

TABLE III

ho

O

TABLE IV

B D E F H K hJ

[0058] As can be seen in Tables III and IV above, in addition to providing kill data, skin irritation test results are provided. Both of the formulations subjected to irritation testing provided good results.

[0059] Table V below shows the use of a variety of other components intended to adapt the product for topical use. For example, some embodiments have shown the inclusion of skin conditioners, such as glycerine and sorbitol. Other embodiments show the inclusion of barrier agents, such as Maltrin M40, Propylene Glycol, PVP K30, and Pluronic 108. Results for viscosities, look, and film qualities are also shown in addition to L/D ratios. Table VI shows that for some formulations using the skin-conditioner sorbitol, significant kill numbers were obtained using a number of well-known barrier agents.

TABLE V

B C D E H

TABLE VI

[0060] It should be recognized that pH as well as Akacid Plus concentrations each play a significant role in kill effectiveness. This is reflected in the charts provided below which reflect kills at 30 seconds (Table VII) and 5 minutes (Table VIII).

[0061] The activity of germicidal compositions was tested by a procedure involving preparing a test tube containing 1 g (10%) or 2 g (20%) of manure, 9.9 mL of germicide and 0.1 mL of ~lxl O⁷ bacteria. The reagents within the test tube were mixed for 30 seconds and then 0.1 mL of the mixture was added to a 6 mL well of a microwell plate containing 0.9 mL neutralizer. After mixing with the neutralizer, 4.5 mL of agar was added to the well. The microwell plate was then incubated for 24 hr to 48 hr. A visual score for the concentration of bacteria was recorded. A score of 5 indicated complete kill of bacteria when no colonies of growth were observed in the well. A score of 0 indicated no kill when numerous colonies of bacteria were observed. The score was determined by a visual comparison to standards which did not contain a germicide (score = 0) or which had no bacteria added (score = 5). Duplicate samples were randomly distributed in the microwell plate so that each visual observation was made twice without knowledge of the identity of the sample contained in each well. This method for the visual determination of sanitizer efficacy has given reproducible results. Although not specifically expressed in the charts, each of the formulation embodiments shown in Tables VII-X included 5% Neodol 91-8.

TABLE VII

TABLE VIII

[0062] As can be seen in Tables VII and VIII above, manipulation of pH to about 8 dramatically increased the kill effectiveness of the Akacid Plus product for different Akacid Plus levels. As will be seen in Tables IV and X below, pH levels around 8 also dramatically improved kill effectiveness at different Akacid Plus concentrations. TABLE IX

TABLE X

[0063] Those skilled in the art will appreciate that the foregoing discussion teaches by way of example, and not by limitation. Insubstantial changes may be imposed upon the specific embodiments that are shown and described without departing from the scope and spirit of the invention.

Claims

CLAIMSWe claim:

1. An antimicrobial composition comprising: a polymeric guanidine salt-based biocidal agent; said composition being adapted for topical usages.

2. The composition of claim 1 wherein said biocidal agent is cationic.

3. The composition of claim 1 wherein said polymeric guanidine salt- based biocidal agent includes one of PoIy-(H exaMethylendiamineGuanidinium hydrochloride) (PHMG) and Poly-[2-(2-Ethoxy)EthylGuanidinium hydrochloride] (PEEG).

4. The composition of claim 3 wherein said polymeric guanidine salt- based biocidal agent includes both PHMG and PEEG salts.

5. The composition of claim 4 wherein a ratio of PHMG to PEEG is about 3 : 1 by weight.

6. The composition of claim 1 comprising a surfactant.

7. The composition of claim 6 wherein said surfactant is an ethoxylated nonionic surfactant.

8. The composition of claim 1 wherein a pH of said composition is adapted to be greater than 8.

9. The composition of claim 8 wherein said pH of said composition is adapted to be between 8 and 10.

10. The composition of claim 9 comprising: a surfactant.

11. The composition of claim 1 comprising one of: a skin-conditioner, a barrier-forming agent, a foaming agent, a viscosity control agent, a pH adjusting agent, a wetting agent, an opacifying agent, and a chelating agent.

12. A method of treating an animal against microbials, said method comprising: including at least one polymeric guanidine salt in a biocidal agent; and adapting said biocidal agent such that said agent is able to be applied topically without said composition causing irritation.

13. The method of claim 12 comprising: using a skin-conditioning agent and a surfactant in executing said adapting step.

14. The method of claim 12 comprising: applying said composition on a bovine.

15. The method of claim 12 comprising: using said composition in a teat-dip application.

16. The method of claim 12 comprising: maintaining a pH of said composition above 8 for the purpose of improving biocidal effectiveness of said composition.