US20120289591A1 - Antimicrobial Compositions Containing Free Fatty Acids - Google Patents

Antimicrobial Compositions Containing Free Fatty Acids Download PDF

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US20120289591A1
US20120289591A1 US13/510,361 US201013510361A US2012289591A1 US 20120289591 A1 US20120289591 A1 US 20120289591A1 US 201013510361 A US201013510361 A US 201013510361A US 2012289591 A1 US2012289591 A1 US 2012289591A1
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acid
free fatty
composition
lecithin
caprylic
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Michael Anthony Folan
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/04—Dispersions; Emulsions
    • A61K8/06—Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/361—Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/55—Phosphorus compounds
    • A61K8/553—Phospholipids, e.g. lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients

Definitions

  • This invention relates to antimicrobial compositions containing free fatty, acids and to pharmaceutical formulations containing same.
  • Free fatty acids are essentially insoluble in water. Their insolubility and the fact that they are incompatible with many conventional excipients have seriously restricted their medicinal use to, date. While salts of free fatty acids are soluble in water, they are known to have greatly reduced antimicrobial effect.
  • Several methods have been used to ‘solubilise’ free fatty acids, including the use of alcohols and surfactants and derivitisation by re-esterification to form mono-glycerides and/or ethoxylation and propoxylation procedures.
  • WO 03/018049 discloses the antimicrobial activity of milk serum apo-proteins in combination with free fatty acids from milk fat. It is illustrated that the adhesion inhibitory properties of milk extracts are exclusively attributable to the water soluble protein fraction and that the lipid component makes no contribution to this effect.
  • WO 2009/072097 discloses properties of compositions of free fatty acids such as melting point depression and sequestration which affect antimicrobial potency. Also disclosed are emulsification methods used to incorporate blends of free fatty acids in a milk whey protein isolate.
  • the invention provides an antimicrobial composition comprising:
  • the free fatty acid may be selected from: butyric (C4:0), valeric (C5:0), caproic (C6:0), heptanoic (C7:0), caprylic (C8:0), pelargonic (C9:0), capric (C10:0), undecanoic (C11:0), undecylenic (C11:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), margaric (C17:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3), arachidonic (C20:4) and euricic (C22:1).
  • the free fatty acid is preferably selected from: valeric, caproic, caprylic, pelargonic, capric, undecanoic, undecylenic, lauric, myristic, palmitic, stearic, oleic, linoleic and linolenic acids and mixtures thereof, and pharmaceutically acceptable salts and esters thereof.
  • Particularly preferred are caproic; caprylic, pelargonic, capric, undecylenic and lauric acids, especially caprylic acid.
  • combinations of fatty acids are, used including higher melting point entities such as lauric acid, lower melting point entities such as caprylic or oleic are preferably included to depress the melting point of the combination to less than normal physiological temperatures.
  • Membrane lipids are ubiquitous components of all cell membranes in the plant and animal kingdoms. They are characteristically made up of one or in most cases two long chain hydrocarbon molecules attached to a highly polar head group, which is a derivative of either, glycerol-3-phosphate, a long chain amino alcohol (sphingosine), a sugar, or a derivative of a steroid (cholesterol).
  • the properties of each membrane lipid are mainly dictated by the variation in the polar head group. Nearly all are amphoteric in so far as they behave as both acid and base and more importantly all are amphipathic, having a water soluble, hydrophilic end at the polar head group and a fat soluble lipophylic end at the hydrocarbon tail.
  • Polar Head Class of Membrane Lipids group Membrane Lipids Lecithin/Phospholipids/ Glycerol Phosphatidic acid Glycerophospholipids Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol phosphatidylinositol Phosphatidylserine Sphingolipids & Sphingosine Ceramide glycosphingolipids Sphingomyelin Glycoglycerolipids Saccharide Glycolipids Glycosphingolipids Cerebrosides Gangliosides Glycoglycerolipids Mono-galactosyl diglyceride Cholesterol Steroids Lanosterol
  • glycerophospholipids those containing phosphate in the polar head group are the most common. Described as glycerophospholipids, phosphoglycerides or more frequently as phospholipids this group makes up the major portion of all membrane lipids in the bacteria, plant and animal kingdoms. All phospholipids are based on a glycerol backbone with two hydrophobic acyl side chains on carbons at sn1 and sn2 and a phosphate moiety at sn3.
  • Variations in the phosphate head differentiate six separate types of commonly occurring phospholipids, the simplest being phosphatidic acid and progressing with increasing complexity through phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, and phosphatidyl inositol, phosphatidylcholine being the most prevalent in animals and bacteria.
  • lecithin a membrane lipid of commerce which is extracted on industrial scale from a variety of sources including but not limited to soya bean, sunflower, canola, palm oil, egg yolk and butterfat.
  • lecithin is frequently used synonymously with the major phospholipid component phosphatidylcholine, which may be purified from crude lecithin, as is usual when rigorous control of quality is required in pharmaceutical applications. It is also possible to enzymatically modify lecithin or any of its component phospholipids by, for example, removing one of the hydrocarbon side chains to form ‘lysophospholipids’ all of which are equally suitable for use in this invention.
  • Hydrolysed derivatives of membrane lipids include lyso-phospholipids and lyso-sphingolipids which may be produced enzymatically using pancreatic phospholipases available from Novozyme, Denmark.
  • the process involves preparation of an aqueous dispersion of phospholipids or sphingolipid, addition of phopsholipase enzyme at 2% W/V and incubation at an elevated temperature of from about 50° C. to about 60° C. for about 2 hours.
  • Yield of hydrolysed membrane lipid may be up to 70%, and the product may be separated from the mixture by water partition based on increased hydrophilicity of the lyso derivative.
  • the membrane lipids used herein may be extracted from plant or animal sources including oil bearing seeds, animal fat, wool, milk and eggs.
  • the membrane lipids and derivatives thereof used in the present invention are preferably delipidised.
  • the term “delipidised” is intended to mean that substantially all of the conjugated extraneous lipid material, such as oil, fat or triglyceride material, with which membrane lipids are normally associated, in nature, is removed. “Substantially all” in this context is intended to mean that the delipidised membrane lipid contains less than 10%, preferably less than 5%, more preferably less than 3%, of conjugated extraneous lipid material.
  • the membrane lipid is preferably selected from one or more of phospholipids, lecithin, glycerophospholipids, sphingolipids, glycosphingolipids, glycoglycerolipids and cholesterols.
  • Suitable membrane lipids include lecithin, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, ceramide, sphingomyelin, glycolipids, glycosphingolipids, cerebrosides, gangliosides, glycoglycerolipids, mono-galactosyl diglyceride, lanosterol or cholesterol or any combination thereof.
  • lecithin and other phospholipids selected from phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylserine and mixtures thereof.
  • Lecithin is particularly preferred.
  • a combination of one or more of caproic, caprylic, pelargonic, capric, undecylenic and lauric acids with one or more phospholipids is particularly preferred, especially caprylic acid with lecithin that is preferably delipidised.
  • the ratio of free fatty acid or pharmaceutically acceptable salt or ester thereof to membrane lipid may be from about 0.25:1 to about 10:1; or from about 0.5:1 to about 10:1, or from about 0.5:1 to about 5.0:1, or from about 1.0:1 to about 2.5:1, or from about 1.25:1 to about 2.5:1, on a weight for Weight basis.
  • the antimicrobial composition of the invention may also comprise one or more pharmaceutically acceptable organic acids or a pharmaceutically acceptable salt or ester thereof; and/or one or more pharmaceutically acceptable inorganic acid salts.
  • the organic acid is preferably selected from acetic, pyruvic, propionic, glycolic, oxalic, lactic, glyceric, tartronic, malic, maleic, ascorbic, fumaric, tartaric, malonic, glutaric, propenoic, cis or trans butenoic and citric acids and mixtures thereof, and pharmaceutically acceptable salts and esters thereof.
  • the inorganic acid salt is preferably selected from chloride, sulphate and nitrate.
  • Particularly preferred organic acids are citric and lactic acids and the sodium and potassium salts thereof, especially sodium citrate.
  • the molar concentration of the organic acid is preferably from about 25 mM to about 500 mM, more preferably from about 50 mM to 250 mM, and more preferably from about 50 mM to 150 mM.
  • the pH of the organic acid salt is preferably between 4.0 and 6.5, preferably between 4.0 and 5.5 or between 4.0 and 5.0.
  • the membrane lipid emulsifies the fatty acid, rendering it water-dispersible.
  • the membrane lipid-fatty acid combination is an emulsion, preferably an oil-in-water emulsion, although a water-in-oil emulsion is also possible.
  • the composition of the invention comprises an emulsion of 0.5% caprylic acid in 0.4% de-lipidised lecithin, which is then diluted to 50% of its concentration using 200 mM sodium citrate buffer at pH 4.5, the final concentration being 0.25% caprylic acid emulsified in 0.2% de-lipidised lecithin and dispersed in 100 mM sodium citrate at pH 4.5.
  • This composition is referred to hereinafter as the “standard formulation” and may conveniently be used to demonstrate the antimicrobial effects of the inventive compositions.
  • the antimicrobial compositions of the invention exhibit a dual antimicrobial effect, in that they exhibit inhibition of microbial adhesion to host cell surfaces and achieve a microbicidal effect through dissolution of microbial cell membranes.
  • membrane lipids particularly phospholipids, collectively described as lecithins
  • Inhibition of microbial adhesion requires that the lipophylic end of the membrane lipid is free to orientate with and conjugate to lipid moieties on the microbial cell surface. This process is impeded if the lipophylic sites are occupied by non-membrane lipids (triglycerides for example).
  • a particular utility afforded by the inventive compositions is the discovery that the different classes and different members of each class of membrane lipids have different release characteristics.
  • the same dose of the same fatty acid in the same amount of different membrane lipid will deliver the same microbicidal effect over a faster or slower period depending on the individual membrane lipid.
  • This peculiar characteristic facilitates the use of combinations of different membrane lipid emulsions of the same fatty acid to achieve a sustained release effect over relatively useful periods.
  • the antimicrobial compositions of the invention can be used to regulate the rate of blood clotting.
  • the formulations will either catalyse the rate of blood clotting or suppress it altogether. This property gives great advantage in use as a blood contact antimicrobial agent.
  • an aqueous dispersion Of de-lipidised membrane lipid added to fresh sheep blood at a volume of 20% will accelerate the normal rate of blood clotting, reducing the time to clot from 6 minutes to less than one minute.
  • Addition of a fatty acid, such as caprylic acid, by emulsification up to a ratio of about 1.0-1.3 times the weight of de-lipidised membrane lipid, such as lecithin will further reduce the time to clot formation, but thereafter as the ratio of fatty acid increases, the time to clot formation is increased and an anti-coagulation effect is observed when the weight of emulsified fatty acid exceeds about 1.0-1.3 times the weight of de-lipidised membrane lipid.
  • the blood regulatory effect will depend not only on the ratio of free fatty acid to de-lipidised membrane lipid, but also on the nature of the membrane lipid and free fatty acid used.
  • an emulsion of free fatty acid not exceeding about 1.0-1.3 times the weight, of de-lipidised membrane lipid may be used to enhance the rate of blood clotting and exert an antimicrobial effect at the site of bleeding, while an emulsion of free fatty acid exceeding about 1.0-1.3 times the weight of de-lipidised membrane lipid with or without added salts of organic acids may be used to inhibit blood clot formation and exert a microbicidal effect at the site of bleeding.
  • compositions of the invention containing membrane lipid emulsified free fatty acids exert vastly superior dual antimicrobial effects: both inhibition of adhesion and microbicidal effect.
  • dual antimicrobial effect is that the adhesion inhibitory properties prevail long after the microbicidal pay-load has been exhausted.
  • Most microbicides are chemically reactive with the target organism and most microbicidal reactions are irreversible under physiological conditions; a fixed dose of a microbicide therefore has a limited reactive capability.
  • the adhesion inhibitory properties persist after the microbicide has been depleted and additional protection is afforded against any residual viable pathogens.
  • compositions of the invention are compatible with systemic administration (blood contact), in contrast to milk protein compositions disclosed in WO 03/018049, and WO 2009/072097.
  • Membrane lipids and free, fatty acids are natural metabolites in the human and animal body, their antimicrobial effects are concentration dependent and when they enter the systemic circulation they are rapidly diluted, metabolized and excreted as natural metabolites.
  • membrane lipids in combination with an antimicrobial agent are counter-intuitive as most conventional antimicrobial agents are inactivated by lecithin and related membrane lipids; lecithin is listed as an approved antiseptic neutralizing agent for use in European Standard EN 1499 and 1500 testing for evaluating the microbicidal effect of hand soaps and gels.
  • compositions of the invention may be used in the treatment or prophylaxis of microbial infections in humans or animals. Due to the regulatory effect on blood-clotting, the compositions may be used in, for example, catheter locking solutions and in wound care.
  • the invention also provides a pharmaceutical formulation comprising a composition according to the invention and a pharmaceutically acceptable carrier, diluent or excipient therefor.
  • the composition may be present in the pharmaceutical formulation in an amount of from about 0.1% to about 25% (w/v); or from about 0.1% to about 10% (w/v); or from about 0.1% to about 1.0% (w/v).
  • the pharmaceutical formulation may be in any form suitable for administration to a human or animal, including for example, forms suitable for oral, topical, enteral, parenteral or mucosal administration.
  • the formulation of the invention can be used as a topical antimicrobial agent for prevention and treatment of infections of the skin, hair, nails and external membranes of the body orifices.
  • the inventive composition or formulation may be used in the construction of a liquid soap or hand gel for elimination of asymptomatic carriage of potentially pathogenic bacteria such as methicilin resistant Staphylococcus aureus and other species commonly associated with nosocomial or hospital acquired infections. It may be used as a gel for treatment of acne. It may be used in the form of an ointment for treatment of dermatophytic fungal infections of the hair follicles including that caused by Trychophyton species commonly known as ringworm. It may be constructed in the form of a spray for delivery to the skin as a treatment for infections caused by the enveloped viruses including Herpes varieties causing cold sores and shingles. It may be prepared in the form of liquid drops for treatment of infections of the eye and ear.
  • It may be prepared in a variety of pharmaceutically acceptable delivery systems for treatment of infections of the mucosa including the mucosal surfaces of the nose, mouth, throat, bronchiole and lungs and the gastro-intestinal tract and genitalia. It may for example be prepared in the form of a toothpaste and mouthwash to facilitate improved dental hygiene and eliminate the burden of organisms causing dental caries, gum disease, aphthous ulcer and halitosis. It may be prepared in a form suitable as a saliva supplement for alleviation of the symptoms of xerostomia. It may be prepared as a spray for de-colonisation of the mouth, throat and nasal membranes, particularly for eradication of asymptomatic carriage of antibiotic resistant species.
  • It may be prepared, in the form of a gel for the prevention and treatment of microbial infections of the genitalia caused by a variety of bacteria, yeast and viruses including those known to be the causative agents of Candidiasis, non-specific bacterial vaginosis, the herpes virus and HIV. It may be prepared in the form of an enema for prevention and treatment of infections of the bowel and lower intestinal tract including those caused by anaerobic Clostridium species and Desulfovibrio medically known as pseudomembraneous colitis and pouchitis.
  • Membrane lipid emulsified free fatty acids may be used to prepare foods that exert an antimicrobial effect in the gastro-intestinal tract, serving to protect against enteric pathogens such as Helicobacter, E. coli, Salmonella, Campylobacter, Clostridium species, Protozoa and enveloped viruses.
  • enteric pathogens such as Helicobacter, E. coli, Salmonella, Campylobacter, Clostridium species, Protozoa and enveloped viruses.
  • a suitable food carrier for membrane lipid emulsified free fatty acids is dairy milk, preferably fat free dairy milk and more preferably skim milk powder such as Marvel, available commercially from Premier International Foods (UK) Ltd, Spalding, Lincolnshire, England.
  • emulsions of, for example, caprylic, capric and lauric acids may be prepared as 5.0% W/V emulsions in 4.0% W/V de-lipidised lecithin as described in the methods.
  • the individual emulsions may be combined in a ratio of 1:1:1 or any other suitable ratio such as 1:2:3.
  • Membrane lipid emulsions of other fatty acids such as butyric and or emulsions of essential oils such as peppermint or vanilla may be added to impart flavour and improve taste.
  • Marvel skim milk powder may be re-constituted by adding the specified amount to potable water (4 heaped teaspoons to one pint). To this an amount from about 1% W/V to 15% W/V of a selected ratio of membrane lipid emulsified free fatty acids may be added and mixed by stirring. Preferably the amount of emulsified fatty acid will be from about 1% W/V to 10% W/V and more preferably about 5% W/V. It will be clear to a skilled person that skim milk powder may be re-constituted in less than the optimal volume of water for use as milk, in which case a concentrated dairy cream is formed to which amounts of emulsified fatty acids may be added.
  • a dairy whey protein isolate may be used as a suitable food carrier: Provon 190 from Glanbia PLC is a suitable whey protein isolate.
  • the whey protein isolate may be re-hydrated in potable water in an amount of from about 10% W/V to 20% W/V, preferably about 15% W/V.
  • an amount of membrane lipid emulsified free fatty acid or blend thereof may be added and mixed by stirring.
  • the amount of added membrane lipid emulsified free fatty acid may be from about 1% W/V to 20% W/V, preferably from about 5% W/V to 15% W/V and more preferably about 10% W/V.
  • the products of the invention have utility as blood contact antimicrobial agents where their combined blood clotting or anti-clotting capability is combined with their antimicrobial effects.
  • Such uses include surgical irrigation, wound care, catheter locking solutions, the coating of catheters and other tubular surgical devices for insertion into a bodily orifice or cavity, and in food safety.
  • a significant advantage of the products of the invention is that very short exposure times are required to achieve an antimicrobial effect, generally less than 1 hour or less than 30 minutes or less than 10 minutes.
  • antimicrobial refers to any substance, component or composition of components which exhibit an antagonistic effect towards protozoa, gram positive and negative bacteria (both, aerobes and anaerobes), yeast, fungi, mycoplasma and/or viruses and in particular those microbial species that are capable of causing disease in humans and animals.
  • Infectious disease arises either from ingress of pathogenic microbial species (microorganisms) from the external environment or as a result of aberrant growth of microorganisms that are normally present in the natural micro-flora of the skin, hair, and mucosal membranes of the eye, nose, mouth, gastro-intestinal tract and the genitalia.
  • pathogenic microbial species microorganisms
  • membrane lipid emulsified free fatty acids present wide ranging utility in human and animal healthcare.
  • these include surgical irrigation fluids, haemostatic antimicrobials in wound care, catheter coatings and catheter locking solutions for prevention of catheter related bacteraemia; in mucosal healthcare they can be used to replicate the natural antimicrobial mechanism of healthy mucus; in topical skin care they can be used to amplify the natural antimicrobial defenses of the skin; in food safety because they are natural metabolites they can be applied directly to a food surface to eradicate food borne pathogens; and as medical foods they can be used to supplement a normal diet to prevent or treat infections of the gastro-intestinal tract.
  • MRSA Methicillin Resistant Staphylococcus Aureus
  • Enterococcus species Pseudomonas
  • yeast Candia albicans the yeast Candia albicans .
  • open wounds are irrigated to clear them of loose tissue, blood and other body fluids and in many cases a solution of sterile saline is currently in use, although this offers no antimicrobial effect.
  • the use of surgical irrigating fluids containing antibiotics is not recommended simply because these have the potential to generate further antibiotic resistant species.
  • compositions based on optionally de-lipidised membrane lipids combined with free fatty acids and/or derivatives thereof as described herein provide such utility. Also in trauma care after accidental injury or during military operations, wounds are frequently jagged, dirty and possibly hemorrhaging profusely. There is a great need for emergency intervention with antimicrobial products that promote blood clotting and which can be applied safely to an open wound.
  • a catheter In simplistic terms a catheter is a tube inserted through the skin, into an artery or vein or through a natural orifice for the purpose of draining body fluids, or for administration of drugs or for the purpose of monitoring disease or manipulation of surgical instruments. Some catheters remain in place for relatively long periods of time and in many cases these are susceptible to microbial contamination causing bio-film on the inside and outside surfaces and potentially leading to disseminated infection of the patient.
  • CVC central venous catheter
  • a catheter locking solution is a volume of fluid sufficient to fill the lumen of the catheter between medical procedures.
  • An optimal CLS will exert an antimicrobial and anti-coagulation effect.
  • the selection of active ingredients to construct these CLS formulations is constrained by the risk of accidental intra-venous infusion.
  • the void volume of a CVC may be as much as 3.0 ml or more. In the event that a medical administrator forgot that a locking solution had been inserted and accidentally added a second dose, the first void volume would be infused directly into the blood stream where it could, have serious toxicological implications for the patient.
  • the formulations herein may be in the form of catheter locking solutions, which can achieve optimal microbicidal effect in less than 1 hour and have anti-clotting properties similar to heparin without the use of that material in the formulation.
  • the catheter locking solutions preferably have a viscosity approximating to that of whole blood, to minimize the potential for dilution and mixing at the catheter tip.
  • the desired viscosity can be achieved using viscosity-enhancing agents.
  • Viscosity-enhancing agents routinely used in pharmaceutical preparations include a wide range of hydrogels of natural or synthetic origin. Included among these are derivatives of cellulose such as carboxymethyl cellulose and hydroxyethyl cellulose. Other natural polymers of plant origin include dextran, alginate, pectin, guar gum and acacia gum; synthetic polymers include a range of carbomers (acrylates/C10-30 alkyl acrylate cross polymers) and Poloxamers (triblock copolymers of polyoxyethylene and polyoxypropylene). For reasons of residual toxicity and metabolic clearance, few of these are approved for routine systemic use in human medicine. The Poloxamer, Pluronic F68 has been used in parenteral nutrition formulations, but the naturally occurring dextran has achieved more extensive use in surgery.
  • dextran is the preferred viscosity-enhancing agent.
  • Dextran is a naturally occurring polysaccharide. A complex branched glucan of glucose monomers, it is produced by many bacteria including Leuconstoc species, and it has been used as an irrigant and anti-clotting agent in micro-surgery. Dextran is available commercially in a range of molecular weights ranging from 10 Kilo daltons (Kd) to 150 Kd. In the invention, 20 Kd to 60 Kd dextran is preferred. More preferably, 40 Kd dextran is used to adjust the viscosity of a membrane lipid/free fatty acid-based catheter locking, solution to approximate to that of whole blood: 3.6-6.5 cP.
  • 40 Kd dextran When 40 Kd dextran is used as viscosity-enhancing agent, its concentration in formulations should preferably be from about 5% to 50% on a weight by volume basis, more preferably from about 10% to 40%, and more preferably still from about 15% to 30% weight by volume based on the entire formulation.
  • compositions of this invention can be used to create dual action anti-adhesion and antimicrobial surface coatings by trapping a film of free fatty acid, on any animate or inanimate surface, including but not limited to skin, plastic, rubber, metal or glass, wherein they exert a microbicidal effect at the surface in addition to repelling adhesion of microbial species.
  • Active antimicrobial surface coatings have particular application in healthcare, for prevention of bio-film formation on medical instruments and on catheter surfaces, and also as a surface coating for work-stations, procedural trays and all patient contact surfaces, including the hands of healthcare workers.
  • active antimicrobial surface coatings have extensive application in the food industry where they can be applied to food preparation and packaging surfaces to minimize carriage of food borne pathogens such as Salmonella, Campylobacter, Listeria and E. coli and as demonstrated herein, they can also be applied to the surface of food products, particularly post-slaughter meal, to eradicate these pathogens at source.
  • an antiseptic wipe which may contain triclosan, chlorhexidine, quaternary ammonium compounds or a concentrated solution of alcohol.
  • an antiseptic wipe which may contain triclosan, chlorhexidine, quaternary ammonium compounds or a concentrated solution of alcohol.
  • the residual toxicity of many conventional antiseptics limits their use on food and animate surfaces.
  • composition of the invention When used in surface coating applications, the composition of the invention may be applied in a suitable pharmaceutically acceptable delivery system, with or without other excipients.
  • suitable pharmaceutically acceptable delivery systems include, but are not limited to, organic solvents designed to evaporate on application leaving a dry residue, liquids, creams, gels, pastes, ointments, powders or sprays and include combinations of these with insoluble materials such as fibrous wipes.
  • the mucus membranes of humans and animals are characteristically moist surfaces at the interface of natural body orifices, and the lining of the gastro-intestinal tract and the genitalia, they include the eye, nose, inner ear, mouth, naso-pharyngeal surfaces, trachea, bronchioli, esophagus, stomach, large and small intestines, rectum, vagina and external labia, the glans and the lining of the urinary tract.
  • mucosal structures that are unique to particular species such as the guttural pouch in equids.
  • Mucus membranes are covered by a layer of mucus, a viscous hydrocolloid comprised mainly of mucins, a group of heavily glycosylated high molecular weight proteins which act as a matrix within which many other biologically active materials are dispersed including secretory antibodies and components of the innate immune system such as histatins and statherins in saliva.
  • mucus is essential for prevention of ingress of potential microbial pathogens and for prevention of adhesion and colonization of the mucus membranes themselves.
  • Impairment of mucosal secretions and debilitation of the integrity of the mucus itself may arise as a consequence of disease, or as a side effect of medical and/or pharmaceutical intervention or as a consequence of life-style. Where this happens, those afflicted suffer from recurring mucosal infection including but not limited to dental caries, gum-disease, oral thrush, yeast vaginitis, bacterial vaginosis, enteritis and infectious colitis.
  • the complexity of mucus has defied all attempts to construct exact replicas which may be used in replacement therapy. There are a number of commercial substitutes, which are designed to relieve the pre-dominant physical symptoms of oral dryness.
  • the skin, hair and nails of mammals, being the external body surfaces are subject to constant environmental contamination. Healthy mammalian skin has intrinsic antimicobial properties based on naturally occurring free fatty acids in the skin, and these may be advantageously fortified by topical application of delipidised membrane lipid-emulsified free fatty acids as disclosed herein.
  • Bacteria causing skin infection include, but are not limited to, Staphylococcus aureus, Streptococus pyogenes and Pseudomonas areruginosa , causing Impetigo, Folliculitis, Erysipilis, Cellulitis and Necrotising Fasciitis.
  • Propionibacteriium acne is the causative agent of juvenile acne.
  • Fungal skin infections are caused mainly by Trichophyton, Microsporum and Epidermophyton species and the diseases are known collectively as Tinnea (pedis, cruris, capitis, corporis and unguinium) which include ringworm and nail infections.
  • Enveloped viral infections include Herpes Simplex Type 1 affecting orofacial regions and type 2 affecting the genital regions. Herpes zoster causes shingles and the poxvirus causes Molluscum contagiosum. Superficial asymptomatic carriage of HIV, SARS, Hepatitis, Swine flu, bird flu and many other zoonotic viral infections may also be eliminated using the compositions herein.
  • HAI hospital acquired infection
  • MRSA Methicilin Resistant Staphylococcus aureus
  • VRE Vancomycin Resistant Enterococci
  • Clostridium difficile and other less well known opportunistic pathogens including Pseudomonas and Candida.
  • Food borne pathogens include Salmonella, Escherichia, Campylobacter and Clostridium species.
  • the efficacy of the products of this invention in vitro is demonstrated in the Examples.
  • Methods of eradication of food borne pathogens at primary processing of meat include the proposed use of carcass washing with a suitable antimicrobial agent.
  • Other methods of application that are relevant to food safety include the use of membrane lipid-emulsified free fatty acids as surface coatings for food packaging using procedures similar to those described for application of antimicrobial surface coatings to medical devices.
  • Free fatty acids greater than C6 are insoluble in aqueous media.
  • Membrane lipids are equally insoluble in water but may be dispersed therein, and under appropriate conditions combinations of free fatty acids and membrane lipid may be induced to form emulsions in water. These are either oil-in-water or water-in-oil emulsions, the latter having the higher relative concentration of fatty acid by weight.
  • a water-in-oil (free fatty acid) emulsion may be prepared by dissolving e.g. about 1 gram of membrane lipid, such as optionally delipidised lecithin, in about 2 grams of free fatty acid. Stir for about 10 minutes and then add about 2 grams of sterile distilled water, agitate vigorously by shaking for 30 seconds and allow to hydrate for 30 minutes. When fully hydrated the emulsion has a thick creamy consistency which may be further diluted with up to 20 ml of water, added in 5 ml aliquots with intermediate agitation by shaking. An excess of water added to this emulsion will cause it to break and invert in part or in whole and become unsuitable for further processing.
  • membrane lipid such as optionally delipidised lecithin
  • Oil-in-water emulsions are better suited to high water content applications such as wound dressings, catheter locking solutions and surface disinfection, as described hereinafter.
  • Methods of preparing oil-in-water emulsions are well known to those skilled in the art and are disclosed in for example, WO2009/072097.
  • a suitable example of an oil-in-water emulsion of free fatty acid in membrane, lipid may be prepared by suspending a suitable quantity of membrane lipid, such as optionally delipidised lecithin, in sterile distilled water, and stirring this at room temperature for about 30 minutes with a magnetic stirrer to ensure even dispersion.
  • a suitable dispersing device the desire amount of free fatty acid, such as caproic, or caprylic, or pelargonic acid, is slowly added while vigorously agitating the volume of dispersed membrane lipid.
  • the amount of membrane lipid e.g. lecithin
  • the amount of membrane lipid is conveniently 0.4 g in 100 ml water, to which 0.5 g of free fatty acid can be added using e.g.
  • emulsion is a thin white liquid suspension of free fatty acid, which is stable and can be diluted many-fold without de-stabilising the emulsion.
  • a similar procedure may be used to prepare an oil-in-water emulsion of, for example, capric acid or undecylenic acid, provided the constituents are first equilibrated at 37° C., or above their melting point. Once emulsified, the emulsion remains stable on cooling below the melting point of the incorporated free fatty acid and may be diluted further for use in formulation.
  • a membrane lipid emulsion of lauric acid may be prepared. in a similar manner provided all constituents are first equilibrated at 50° C. or above the melting point of lauric acid, and again this emulsion will remain stable on cooling and in further dilution for use in formulation.
  • Free fatty acids have limited microbicidal effect at temperatures below their individual melting point. This fact has little significance for those acids with melting points below normal physiological temperatures (37° C.), except in situations where such emulsions may be required to exhibit an effect in hypothermic conditions.
  • Blends of high and low melting point free fatty acids have depressed melting points due to the solvent effect of the lower melting point constituent.
  • a combination of lauric acid with a melting point of 44° C. and caproic acid with a melting point of ⁇ 3.4° C. in a 50:50 ratio will exhibit a combined melting point of 26° C., and the blend may be emulsified as described above at temperatures above 26° C.
  • Higher melting point free fatty acids may also be dissolved in low melting point blends of essential oils or other neutral oils to achieve a depression of melting point suitable for antimicrobial use under physiological conditions.
  • Essential oils are hydrophobic constituents extracted from many different plants by distillation or solvent extraction. They are widely used commercially as perfumes and in fringe medical practice such as aromatherapy. Most notable examples include oil of clove, orange, lemon, lavender, juniper, and rose. Many of these are known to exhibit microbicidal effect in their own right; oil of lemon balm for example is recognized as being an effective viricidal agent and when used as a solvent for higher melting point fatty acids such as lauric acid, the combined microbicidal and viricidal properties provide expanded utility in medical applications.
  • Oil of lemon balm will dissolve up to 40% by weight of lauric acid at 20° C. and the blend may be emulsified in membrane lipid such as lecithin, as described above.
  • Oil-in-water emulsions of free fatty acids or blends thereof may be prepared in concentrations of optionally de-lipidised membrane lipid ranging from about 0.1% to 10% and emulsified free fatty acid loadings of from 01 to 10%.
  • the loading may conveniently be 4% membrane lipid such as lecithin, and 5% free fatty acid or blend thereof. More preferably still a loading of 0.4% optionally de-lipidised lecithin and 0.5% free fatty acid or blend thereof is used, which may be diluted further for use in formulation.
  • microbicidal efficacy of any membrane lipid emulsion of free fatty acid according to the invention is amplified by increasing surface area of the emulsified droplet relative surface area is increased by decreasing the droplet size.
  • Droplet size is a function of the amount of energy imparted by the dispersing tool during the emulsification procedure. Generally, finer droplets may be produced using higher homogenization speeds and the use of an ultrasonic probe adjacent to the emulsification head will also facilitate smaller droplets and increased surface area.
  • De-lipidisation can be achieved by suspending the membrane lipid or hydrolysed derivative thereof in a polar solvent, such as an alcohol or ketone, at a concentration of about 10% w/v and stirring for about 30 minutes during which time extraneous lipid is dissolved.
  • a polar solvent such as an alcohol or ketone
  • Membrane lipids are insoluble in polar solvents and will readily settle out after stirring allowing the polar solvent with its dissolved lipid to be decanted. Residual solvent is allowed to evaporate in e.g. a fume hood.
  • Suitable solvents include acetone and ethanol.
  • a suitable model of microbial adhesion and inhibition of adhesion is provided by the interaction between the yeast Candida albicans and human Buccal Epithelial Cells freshly harvested from the inside the cheek.
  • a standardized population of fresh late log-phase Candida are exposed to a variable concentration of test substance for 10 minutes, then combined with a fixed ratio of buccal epithelial cells (BECs) for 60 minutes, during which time yeast will adhere to the BECs to a greater or lesser degree depending on the potency of the inhibitory substance used in pre-treatment: After the adhesion period, the combined yeast and BECs are diluted by a factor of 2 ⁇ in sterile buffer and agitated briefly (5 seconds) on a laboratory vortex. Wet mount samples of the mix were examined under a microscope using 400 ⁇ magnification.
  • BECs Yeast cells adhering to BECs are clearly visible and enumeration of these is facilitated using dark field conditions; the use of a Neubauer hemocytometer slide facilitates counting. In total 100 BECs are evaluated and the total number of yeast adhering to these is used as the sample count. The control is equivalent to 100% adhesion and reduction in this number in response to test item or protein blank is reported as percent inhibition of adhesion.
  • Buccal epithelial cells are harvested from the inner cheek mucosa of volunteers by rubbing a sterile tongue depressor in a circular fashion and rinsing the collected cells into 5 ml of sterile phosphate buffered saline(PBS). BECs should preferably be collected in the morning before eating or brushing teeth. Donations from 4-5 volunteers are needed to achieve five assay points.
  • BECs are pooled and counted using direct microscopic count with a hemocytometer slide; a count of 500/ml (for example) may be achieved.
  • the pooled sample is centrifuged at 1,500 RPM for 3 minutes, and re-suspended in a volume of sterile PBS, calculated to achieve a concentration of 500 BECs per ml.
  • This concentrate was divided into 2.5ml aliquots in 10 ml centrifuge tubes and centrifuged again at 1,500 RPM for 3 minutes. The supernatant is discarded and the cell pellets held on ice pending their use in the rest of the assay.
  • the yeast used in these assays is a fresh clinical isolate of Candida albicans ; ATCC 10231 may be used as an alternative, but all culture collection ‘Type’ strains have lost some virulence and do not adhere as well as fresh isolates.
  • Yeast cells are maintained on yeast extract peptone dextrose (YEPD) agar, and a loop-full of a pure culture is used to inoculate a 250 ml Erlenmeyer flask containing 100 ml of sterile YEPD broth. The inoculated flasks are incubated overnight (10-14 hours) at 37° C. in an orbital incubator at 100 RPM.
  • YEPD is: 2% W/V glucose, 1% W/V Yeast Extract, 1% W/V bacteriological peptone, Agar where required at 1-2% W/V, all from Oxoid, UK.
  • the late log-phase cells are counted with a hemocytometer slide and the concentration adjusted to 100 ⁇ the test concentration of BECs (40,000/ml in these examples), using 50 mM sodium lactate buffer pH 4.5.
  • 50 mM sodium lactate buffer pH 4.5 2.5 ml aliquots of the standardized suspension of yeast are washed once by centrifugation with sodium lactate buffer at 4,000 RPM and held as a pellet pending the assay procedure as described below.
  • a solution of test substance and protein blank at the required concentration is prepared in 50/100 mM sodium lactate buffer pH 4.5 or sodium citrate buffer pH 4.5; unless otherwise stated, citrate or lactate buffers on their own were used as controls to determine full adhesion.
  • Bovine serum albumin (BSA) is used as a protein ‘blank’, Sigma-Aldrich A-7030; St Louis, Mo., USA.
  • the material is crude, “initial fractionation by heat-shock” as non-heat shocked fractions may contain active serum immunoglobulin which may contribute to the inhibition of adhesion.
  • Washed yeast pellets from above are re-suspended in 2.5 ml volumes of test, blank or control solutions and held at 37° C. for 10 minutes pre-treatment. Each 2.5 ml volume of test, blank or control (with yeast in suspension) is then used to re-suspend a pellet of washed BECs from above. The combined suspensions are then incubated with gentle agitation at 37° C. for 60 minutes. The use of an orbital incubator at 50 RPM provides a suitable environment.
  • the assay may be performed as a pre-treatment of BECs by reversing the order of re-suspension described above.
  • BECs are first re-suspended in 2.5 ml volumes of control buffer, test or protein blank, held at 37° C. for 10 minutes and then used to re-suspend a pellet of washed yeast and the procedure completed as described above.
  • the assay is a standard microbiological suspension test wherein known concentrations of late log phase bacteria, yeast or fungi are inoculated into a fixed, volume or weight of a test substance, blank or control. After a set period of time a neutralizing solution is added to stop the antimicrobial effect and the residual population of viable microorganisms is enumerated by serial dilution and plate counting.
  • the counting procedure is a standard and basic microbiological procedure for enumerating viable microorganisms and will be well known to those skilled in the art.
  • the method requires inoculation of 1 gram or 1 ml of test sample with 0.1 ml of 18 hour (late log phase) followed by vigorous agitation to mix. After the pre-determined exposure time has elapsed, 9.0 ml of neutralizing buffer is added and mixed. This has the effect of stopping the microbicidal effect which allows reliable estimates to be made of the percentage kill achieved by a particular test sample in the period between inoculation and neutralization. Typically exposure periods will range from 30 seconds up to 30 minutes and may progress to several hours where that time period is required to measure the effect. In order to enumerate residual viable cells and from that to compute percentage kill, serial dilutions and plate counts are carried out on the 10 ml sample plus neutralizing buffer.
  • stocks of bacteria, yeast and fungi are routinely stored on beads in 50% glycerol at ⁇ 80° C.
  • small aliquots from these stocks are spread on an appropriate nutrient agar, grown and sub-cultured to ensure purity.
  • 250 ml Erlenmeyer flasks containing 100 ml of broth are inoculated with a transfer loop from pure agar cultures and incubated under constant agitation in a rotary incubator at 37° C.
  • Bacteria are routinely cultured using brain heart infusion (BHI) broth and agar or tryptone soya agar or broth (TSB), both of which may be acquired commercially from Oxoid, UK.
  • TSB has the following constituents: 1.5% W/W tryptone (pancreatic digest of casein), 0.5% W/W peptone (papaic digest of soybean meal), 0.5% W/W sodium chloride, and agar at 1.5% W/W when required as a solid medium.
  • Yeast are grown on YEPD agar or broth (see adhesion assay method hereinabove).
  • the diluting and neutralizing buffers used in the present method are phosphate buffered saline (PBS), containing 137 mM sodium chloride, 2.7 mM potassium chloride and 10 mM phosphate, to which is added 3% polysorbate Tween 80 (anionic surfactant), 0.3% lecithin, and 0.5% histidine as neutralising agents.
  • PBS phosphate buffered saline
  • polysorbate Tween 80 anionic surfactant
  • 0.3% lecithin 0.5% histidine as neutralising agents.
  • neutralising agents are those prescribed under EU Guidelines for ISO certification of microbicidal efficacy and were validated as suitable for neutralizing free fatty acids and chorhexidine at the concentrations used herein.
  • Clostridium difficile for example is an anaerobe and also aero-intolerant dying off quickly in the presence of oxygen.
  • Fungal pathogens such as the Trychophyton species grow in hyphal mode and cannot be enumerated using the standard serial dilution procedure.
  • agar dilution techniques were used and plate culture evaluated to find the minimum inhibitory concentration, i.e. that concentration of product above which no growth was observed.
  • the technique requires preparation of 10 ⁇ concentrations of the test formulation for dilution in 9 volumes of agar.
  • a 4 ml aliquot of this 10 ⁇ concentrate was added to 16 ml of cooled sterile agar and dispensed to a Petri dish—this represented the full strength (100%) formulation in the agar medium, which may then be expressed as the percentage concentration of microbicidal fatty acid in agar.
  • Further dilutions of the 10 ⁇ concentrate with sterile distilled water and the use of 4 ml aliquots of these dilutions in 16 ml of cooled agar allowed the preparation of a series of decreasing concentrations of the product in agar.
  • the test organisms were inoculated onto these agars with a sterile loop and the minimum inhibitory concentration was determined as the lowest dilution of test item at which no growth was detectable.
  • Bio-film is an attachment and accretion of planktonic bacteria, yeast and/or fungi to solid surfaces.
  • the attachment is usually facilitated through exo-polysaccharides secreted by the microorganisms and studies suggest that it happens more easily on hydrophobic surfaces, such as plastic, and less easily on hydrophilic surfaces such as steel.
  • the formation of bio-film is highly significant in medical science, particularly its formation on the surface of indwelling catheters, where it can be the source of catheter-related blood stream infections. Many different microbial species are capable of forming bio-film, in medicine.
  • Staphylococcus aureus Staphylococcus epidermidis
  • Enterococcus faecalis Enterococcus faecalis
  • Escherichia coli Escherichia coli
  • Klebsiella pneumoniae Klebsiella pneumoniae
  • Pseudominas aeruginosa yeast
  • Candida albicans those of greatest significance are Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudominas aeruginosa and the yeast, Candida albicans.
  • the model of biofilm formation used herein is based on the method of Christensen et al. J. Clin. Microbiol. 22: 996-1006, where growth of a selected organism and its biofilm formation in microtitre plate wells is measured by staining with crystal violet and measuring the intensity (Optical Density) of the stained biofilm in a microtitre plate reader at 570 nm wavelength.
  • the intensity of the stain is a measure of the extent of biofilm formation and its reduction in the presence of inhibitory substances is evaluated on the basis of reduction of the stain intensity.
  • the organism used in this assay is Staphylococcus aureus RN4220, a restriction deficient variant derived originally from NCTC 8325 known for its avidity in biofilm formation, particularly in the presence of excess levels of sodium chloride which is added to the culture media to promote that feature, culture media is 3.7% BHI from Oxoid UK to which 4% W/V NaCl is added.
  • Nunc microtitre plate wells (Nunc International, Rosskilde, Denmark) are coated with the test sample and 1:100 dilutions of late log phase bacteria in fresh saline supplemented BHI are dispensed to each test well and incubated for 24 hours to facilitate biofilm formation.
  • the weirs are rinsed three times with sterile distilled water, and dried for one hour at 60° C. to fix the adhering biofilm. Staining is achieved using a 0.4% solution) of crystal violet which is added to each well and agitated thereby rocking the plate for 4 minutes. Excess dye is removed and the plates are rinsed three times with sterile distilled water and dried. When dry, the intensity of the stain in each well is measured using a ASYS Hitech UVM-340 plate reader at 570 nm (ASYS, Eugendorf, Germany).
  • Hemostasis is the inherent ability of the blood to react to traumatic damage by forming a clot to plug the wound and so prevent excessive loss of blood and facilitate tissue repair and healing of the wound. Hemostasis also refers to the inherent ability of the blood to remain liquid in undamaged blood vessels and so fulfill its primary physiological transport functions. Both of these hemostatic aspects can become defective in disease and both can be overwhelmed by traumatic damage, either accidentally or by default during surgical intervention.
  • the ability to manipulate the hemostatic mechanism by either amplifying the rate of clotting to prevent blood loss after accidental trauma or to prevent clotting during surgical repair and/or to prevent clot formation on, or in, surgically inserted medical instruments including catheters, is of great importance in medicine.
  • To measure the rate of blood clotting herein use is made of a whole blood clotting meter, the Hemochron Signature 11, manufactured by ITC, International Technidyne Corporation, Edison, N.J., USA.
  • the meter uses customized cuvettes into which one drop of fresh blood may be added and the time taken for clot formation is measured optically and reported electronically by the device.
  • Comparative measurement of accelerated clotting times is achieved using appropriate volumes of fresh blood mixed with a volume of test material, where the combined volumes totalled 5 ml in each case in a 15 ml graduated Greiner centrifuge tube (Greiner Bio-one, North Carolina, USA). Immediately after addition of fresh blood, the tubes are inverted twice to mix and allowed to stand without further agitation. Clot formation is visibly apparent when the tubes are tilted slightly, and once a clot formed its integrity was such that it would remain suspended when the tubes were inverted. In each test a 5 ml sample of fresh whole blood (undiluted) is used as a control.
  • Routine analytical High Performance Liquid Chromatography is a suitable method of analyzing the individual membrane lipid components in a composition such as lecithin.
  • the procedure is well known to those skilled in the art of analytical chromatography.
  • a Waters 2420 ELSD HPLC system from Waters Corporation, Milford, Mass., USA is used herein.
  • a Symmetry C8 column (3.0 ⁇ 150 mm, 5 micron column is suitable with a non-linear gradient of 82% methanol in water containing 0.1% tri-fluoroacetic acid and gives adequate separation of membrane lipid components.
  • the reduction in viability of mammalian cells in the presence of membrane lipid emulsified free fatty acids was evaluated using Raji B lymphocytes grown in RPMI 1640 media containing 10% fetal calf serum and Gibco Penstrep 15140 antibiotic supplement. Mature cells are harvested by centrifugation at 1,000 RPM for 3 minutes and re-suspended in RPMI 1640 without supplements for test purposes. Toxicity is assessed by evaluating uptake of trypan blue dye by dead cells using an Invitrogen Countess Automated Cell Counter (Invitrogen Inc, Carlsbad, Calif., USA). The procedure involves exposing a population of Raji B cells to the test solution by mixing 100 ⁇ l of test and cell suspension in a microtitre plate well.
  • FIG. 1 illustrates the antimicrobial inhibitory effect of blood serum and the counteraction of this effect in combination with salts of organic acids, as described in Example 3.
  • FIG. 2 illustrates blood clotting and anti-dotting properties of varying components of a product according to the invention, as described in Example 5.
  • FIG. 3 illustrates anti-clotting properties of a catheter locking solution according to the invention, as described in Example 7.
  • FIG. 4 illustrates comparative microbicidal potency of a catheter locking solution prepared according to the invention and compared with alternative commercial products, as described in Example 7.
  • FIG. 5 illustrates the biofilm inhibitory properties of a product according to the invention, as described in Example 8.
  • FIG. 6 illustrates the variable release of microbicidal effect achieved using different membrane lipids and caprylic acid products according to the invention, as described in Example 9.
  • FIG. 7 illustrates reduction in viability of Candida albicans in response to tailored release characteristics of products according to the invention, as described in Example 10.
  • FIG. 8 illustrates inhibition of adhesion of Candida albicans by tailored release products according to the invention, as described in Example 10.
  • FIG. 9 illustrates the eradication of Staphylococcus aureus and Streptococcus pyogenes from an ex-vivo wound model, as described in Example 12.
  • FIG. 10 is a comparison of the ex-vivo efficacy of a product of the invention in sodium citrate with conventional wound care antimicrobials, chlorhexidine and silver sulfadiazine, as described in Example 12.
  • FIG. 11 illustrates comparative eradication of established biofilm and the superior efficacy of a product of the invention over existing commercially available catheter locking solutions, as described in Example 13.
  • the membrane lipids used in the Examples are delipidised and contain less than 3% conjugated extraneous lipid material as shown by HPLC analysis as described in the Methods.
  • membrane lipids were purchased from Sigma Aldrich, Poole, Dorset, UK.
  • Crude lecithin was purchased from GR Lane Ltd, Gloucester, UK.
  • Aqueous dispersions of each of the above membrane lipids were prepared by suspending 1.0 grams in 100 ml of sterile distilled water (10 mg/ml) and agitating therein for 60 minutes with the aid of a magnetic stirrer. Dispersion of ceramide, sphingomyelin and lanosterol was achieved with the aid of sonication and stirring, while dispersion of all other membrane lipids was relatively easily achieved with stirring alone. Phospholipid and glycoglycerolipid dispersions were relatively stable, but others had a tendency to separate on standing and so all, dispersions were subjected to 30 seconds homogenization using a laboratory homogenizer immediately prior to, use in the adhesion inhibitory assay.
  • a suitable homogeniser is an Ultra-Turax Model T18 (IKA Works, Wilmington, N.C. 28405, USA) fitted with an S18N-19G dispersing tool, operating at 6,000 to 8,000 RPM.
  • each phospholipid was tested as described in the methods at concentrations of 10, 5, and 2.5 mg/ml; the lower dilutions were achieved by diluting the original dispersion with sterile distilled water. Due to logistical limitations of collecting sufficient buccal epithelial cells at any one time, the assays were conducted in blocks of five individual membrane lipids on separate days, and the results shown in Table 4 are a composite of these; the Control is zero concentration of test item and Bovine Serum Albumin (BSA) is used as a protein blank.
  • BSA Bovine Serum Albumin
  • the delipidised membrane lipids were analysed by HPLC as described in the Methods, and all were shown to contain less than 3% conjugated extraneous lipid material. The results are shown in Table 5 below, including the incremental increase in adhesion inhibitory effect achieved by de-lipidisation of individual membrane lipids.
  • Solvent de-lipidisation increases the adhesion inhibitory properties of membrane lipids, especially lecithin by a factor of 2.5, bringing it in line with that of its individual components: PTC, PTE, PTG, PTI and PTS. Further de-lipidisation of the individual components of lecithin achieves further improvement in the adhesion inhibitory properties of these, although less dramatic compared to the effect achieved in de-lipidising crude lecithin. It is assumed here that the commercial process of isolation used to extract individual components of lecithin results in almost complete de-lipidisation, hence the optimal inhibitor effect of these relative to the ‘crude’ lecithin and the smaller incremental effect achieved by additional de-lipidisation.
  • the adhesion inhibitory substance will be in with blood serum, mucus and other body fluids. It has been discovered that de-lipidised lecithin and most of its constituents lose their adhesion inhibitory properties in the presence of Bovine Serum Albumin (BSA). It has also been discovered however, that adding an organic acid salt counteracts this negative effect and restores most of the adhesion inhibitory properties to the combined membrane lipid and BSA.
  • BSA Bovine Serum Albumin
  • 1% solutions of bovine serum albumin i.e. 1% BSA in 200 mM sodium salt of citrate or lactate at pH 4.5.
  • the salt solutions were used to dilute a 1% suspension of de-lipidised lecithin (DLL) and a 1% suspension of phosphatidyl choline (PTC), to achieve preparations of 0.5% DLL and 0.5% PTC in 100 mM sodium lactate/sodium citrate at pH 4.5 with 5% BSA in each.
  • DLL de-lipidised lecithin
  • PTC phosphatidyl choline
  • microbicidal effect of 0.5% caprylic acid in 0.4% de-lipidised lecithin, prepared as described in the Methods, against a range of microbial species may be demonstrated using the microbicidal suspension test described in the Methods.
  • the potency of this material is such that complete eradication of an inoculum in excess of 6 logs may be anticipated in less than 6 minutes.
  • Gram negative species are slightly more resistant than gram positives, particularly those known to be slime producers, such as Escherichia coli and Pseudomonas fluorescens , where it is though that the slime layer protects from contact with the fatty acid for some additional minutes.
  • Table 7 illustrates the reduction in viability of a range of bacteria and yeast. Because the inoculums vary, the latest time to achieve 50% in viability of the inoculum is provided in the last column (L. T. 50%), as a comparative measure of potency against that particular species.
  • MIC value is the % of Caprylic acid in the test plate Organism
  • Organism Notes MIC Clostridium perfringens G ⁇ ve anaerobe >0.1 ATCC 43150 Clostridium difficile G ⁇ ve anaerobe >0.1 ATCC 43598 (aero intolerant)
  • E. coli was selected for this Example as it has been demonstrated to be one of the most resistant species.
  • the bacterium was cultured in Brain Heart Infusion broth for 18 hours at 37° C. 10 ml fractions of each test sample were inoculated at time zero with 1.1 ml of the overnight culture, and 1.0 ml samples removed from this at 3 minute intervals for residual viability determination using serial dilution and plate counting procedures.
  • Example 1 cm 3 sections of fresh beef were deliberately contaminated with late log phase cultures of Salmonella enterica and Escherichia coli O157:H7 at room temperature and allowed to adhere there for 60 minutes. Confirmation of contamination was obtained by mechanically macerating the meat sections in sterile phosphate buffered saline (PBS) and enumeration by serial dilution and plate counting.
  • PBS sterile phosphate buffered saline
  • a suitable but not optimal carcass wash was prepared using 0.5% caprylic acid in 0.4% de-lipidised lecithin and diluting that ⁇ 2 with 200 mM sodium citrate at pH 4.5 as described in Example 3.
  • the wash was sprayed onto contaminated sections of fresh beef, and the antimicrobial effect evaluated by mechanical maceration, serial dilution and plate counting. The results are presented in Table 10 below:
  • the membrane lipid products used may be tailored to affect the rate of blood clotting in addition to contributing a significant antimicrobial effect.
  • Rate of blood clotting was determined using freshly aspirated sheep blood and apparatus and procedures described in the Methods section. An activated blood clotting meter was used to measure anti-clotting effects and a visual tube comparison to evaluate reduced clotting times.
  • aqueous dispersion of de-lipidised lecithin will amplify 2-5 the rate of blood clotting in a concentration dependent manner.
  • Varying concentrations of dispersions of DLL were prepared by suspending the required weight in a volume of water, stirring for 30 minutes to hydrate and homogenizing the suspension with a laboratory homogenizer.
  • Blood clotting and/or anti-clotting effects were assessed using a 20% ratio of test item to fresh blood: in practice 4.0 ml of fresh sheep blood was added to tubes containing 1.0 ml of test item, inverted twice to mix and then either left standing for visual assessment of reduced clotting time or a single drop was applied to the cuvette of an activated blood clotting apparatus for assessment of anti-clotting effect (extended time to clot formation).
  • the observed rate of blood clotting increases, i.e. time to clot formation decreases from 360 seconds for whole blood to approximately 60 seconds or less at DLL concentrations in excess of 1%.
  • caprylic acid by emulsification in DLL will also amplify the dotting effect up to a point Where the concentration by weight equals or slightly exceeds the concentration of DLL by weight.
  • the clotting time is approximately 125 seconds. Addition of emulsified caprylic acid up to 0.5% does not significantly affect the dotting time. Between 0.5% and 0.75% caprylic acid there is a further depression of clotting time from 125 seconds to approximately 40 seconds (70% less). Thereafter, however, the anti-clotting effect is reversed and clotting time increases with increasing concentration of caprylic acid.
  • a suitable, example of an antimicrobial membrane lipid preparation with enhanced clot forming properties may be selected from the combinations prepared in Example 3.
  • a 0.5% dispersion of DLL in sterile distilled water with 1.0% caprylic acid was used here. It will be noted that no organic acid salt is included in this example and further noted that the absence of such reduces the antimicrobial potency but does not limit the clotting effect.
  • the use of a relatively high caprylic acid load compensates for the interference of blood components with the antimicrobial effect.
  • a bacterial inoculum of E. coli was grown in Brain Heart Infusion broth for 18 hours and a 10ml volume of this was sedimented by centrifugation at 4,000RPM for 10 minutes, the pellet was re-suspended in 1.0 ml of the supernatant (concentration ⁇ 10).
  • the BSA blank contained 6.4 ⁇ 10 7 viable E. coli cells per ml, the control blood (heparin/streptokinase treated) sample. contained 8.3 ⁇ 10 5 , and no viable bacteria were recovered from the test sample.
  • preparations such as described in this Example may be applied to wounds in the form of a liquid, spray, gel, powder, or wet-bandage. It will also be clear to those skilled in the art that the preparations described may be added to other pro-coagulants such as chitin, kaolin or alginate to enhance their pro-coagulation effect and add a microbicidal effect.
  • pro-coagulants such as chitin, kaolin or alginate to enhance their pro-coagulation effect and add a microbicidal effect.
  • Ingress of potentially infectious agents during surgical procedures is a major cause for concern among healthcare professionals.
  • the use of irrigating fluids with antimicrobial effects facilitates prevention of this.
  • a specialized application is the use of anti-clotting liquids to fill the void volume of indwelling catheters during periods when the catheter is not in use.
  • a dispersion of 0.4% de-lipidised lecithin (DLL) with 0.5% caprylic acid emulsified therein is prepared as described in the Methods.
  • the emulsion is diluted to 50% of its initial concentration with 200 mM sodium citrate at pH 4.5, the result being 0.25% caprylic acid, 0.2% DLL in 100 mM sodium citrate, or approximately 2.5% W/V sodium salt of citric acid at pH 4.5.
  • Sodium citrate is prepared by adjusting the pH of 200 mM citric acid with 200 mM sodium hydroxide to 4.5; this is not the same as ‘tri-sodium citrate’ which is commonly used as an anti-clotting agent, because not all of the carboxylic acid residues have been salted out.
  • Dextran 40 is used here as a viscosity-enhancing agent where it has been found that 20% W/V inclusion provides a viscosity of approximately 4 cP, the viscosity of human blood being between 3.6 and 6 cP.
  • the emulsified free fatty acid/membrane lipid catheter locking solution used here has the following constituents: 0.2% W/V de-lipidised lecithin; 0.25% caprylic acid; 20% dextran 40, in 100 mM (2.5% W/V) sodium citrate, pH 4.5. Aliquots of this formulation were dispensed to 15 ml Greiner centrifuge tubes in the following amounts: 0, 0.25, 0.5, 0.75 and 1.0 ml amounts. To each of these 5.0, 4.75, 4.5 and 4.25 ml aliquots of fresh sheep blood were added, respectively. Each tube was evaluated consecutively with fresh blood added immediately after it was aspirated.
  • the respective volume dilutions represent 0, 5%, 10%, 15% and 20% by volume of blood.
  • the tubes were inverted twice to mix and a single drop added to the test well of a Hemochron Signature Activated Blood Clotting Meter.
  • the Hemochron blood clotting system relies on a clot activation process which accelerates time to clot formation; whole blood without additives clots in less than 200 seconds in this apparatus.
  • the timelines in this experiment are not directly comparable therefore with clotting times reported in Example 5 where the baseline for normal clot time is shown as 360 seconds being the observed time for normal (non-activated) clotting.
  • the Hemochron meter goes ‘out of range’ at 1,000 seconds of Activated Clotting Time and more extended time recordings are not available.
  • the inventive product of this Example is better than 25,000 I.U. of Heparin and considerably better than Duralock, Zuragen or Taurolock. It should be noted again however, that these are ‘Activated’ clotting times. In practice, none of the treated samples—apart from control PBS—showed any visual sign of clotting even after several hours.
  • Example 6 The antimicrobial effect of the formulation in this Example was tested using procedures similar to those used in Example 6, with the exception that the clot disrupting agents, heparin and streptokinase, were used to break the clots in the control untreated bloods.
  • the above catheter locking solution according to the invention exhibits significantly better anti-clotting effects and much greater microbicidal effect than existing conventional products.
  • a suitable method of applying a persistent coating of a product according to the invention involves emulsification of 1% caprylic acid in 0.8% de-lipidised lecithin prepared as described in the Methods.
  • An equivalent volume of 100 mM sodium citrate buffer pH 4.5 is added to the emulsion to obtain a final concentration of 0.5% caprylic acid, 0.4% de-lipidised lecithin in 50 mM sodium citrate.
  • Eight Volumes of absolute ethanol are then added slowly to two volumes of the emulsion with constant vigorous stirring to make the final coating material in 80% ethanol.
  • some form of surface conditioning which may include a process known as corona discharge wherein an electrical field is generated across the Surface imparting a residual charge which facilitates adhesion of the applied coat.
  • corona discharge wherein an electrical field is generated across the Surface imparting a residual charge which facilitates adhesion of the applied coat.
  • the ethanol solution described above is sprayed on the surface and dried under forced air conditions at 60° C.
  • coats may be applied to construct a layer of antimicrobial coating.
  • a base layer of membrane lipid may first be applied to an inert surface, and once dried and annealed it is used to ‘anchor’ a second layer of de-lipidised membrane lipid emulsified free fatty acid according to the invention.
  • an organic solvent solution of a membrane lipid is applied to the surface of a microtitre plate well, dried and fixed by heating at 60° C. followed by a further application of an aqueous suspension of caprylic acid emulsified in de-lipidised lecithin prepared as described in the Methods.
  • the de-lipidised lecithin emulsion was dried and annealed to the first lecithin coating by heating at 60° C. for 3 hours. Plates treated with de-lipidised lecithin, without caprylic acid were used as a control and untreated plates were used to determine optimum biofilm formation.
  • a base layer of de-lipidised, lecithin (DLL) was prepared by suspending 5% by weight DLL in 80% ethanol: water, and dispensing 100 ⁇ l aliquots of this to the test wells. Wells for determination of optimum biofilm were left untreated. The ethanol fractions were dried in a fume hood and annealed at 60° C. for one hour in an oven. 1% W/V dispersion of de-lipidised lecithin was prepared in sterile distilled water as_described in Example 1, and a volume of this used to emulsify 0.25% caprylic acid. 100 ⁇ l aliquots of DLL or DLL+0.25% caprylic were transferred to DLL, pre-coated wells and dried in an oven at 60° C. for 3 hours.
  • DLL de-lipidised, lecithin
  • BHI Brain Heart Infusion broth
  • Uncoated planktonic growth is largely unaffected by the coating of inhibitory DLL+caprylic add.
  • Wells coated with DLL alone are slightly reduced, but in comparison, wells coated with the product of this invention (DLL+Cap Inhibited Biofilm) are essentially free of any biofim: the coating itself takes up some of the crystal violet dye which accounts for a small increase in Optical Density in the DLL+Cap wells.
  • Example 10 individual membrane lipids were selected from each of the four classes presented in Table 1 and were the same as those used in adhesion inhibitory studies in Example 1. 0.4% Aqueous dispersions of each were prepared and 0:2% caprylic emulsified in each using procedures described in the Methods for de-lipidised lecithin (DLL). A 0.4% sample of DLL with 0.2% caprylic was also prepared. Each of the membrane lipid preparations was diluted to 50% of its volume with 200 mM sodium citrate at pH 4.5, and therefore each preparation then consisted of 0.1% caprylic acid and 0.2% membrane lipid in 100 mM sodium citrate at pH 4.5. It should be noted here that this is less than half of the microbicidal caprylic-acid content of the test item used in Example 3, Table 9.
  • the yeast Candida albicans was used in this Example, and the inoculum prepared as an 18 hour YEPD broth culture as described in the Methods.
  • the late log phase culture was centrifuged at 4,000 RPM for 5 minutes and re-suspended in one tenth volume of supernatant to concentrate ⁇ 10.
  • Mucosal fortification involves complementary hydration, lubrication and enhanced antimicrobial effect of mucosal secretions of the eye, nose, mouth, naso-pharyngeal surfaces, the gastro-intestinal tract and the genitalia.
  • This Example describes a preparation suitable for fortification of the mucosal secretions of the mouth and vagina and most particularly suitable for use by individuals susceptible to recurring oral and/or vaginal thrush and other common bacterial and viral infections responsive to the products of this invention.
  • PTC+Cap PTC+Caprylic acid
  • SGM+Cap SGM+Caprylic acid
  • a test combination preparation comprising fast and slow acting emulsions was prepared by mixing 30% PTC+Cap with 70% SGM+Cap and combining this with an equal volume of part C (30:70 blend).
  • the 30:70 blend is an emulsion of 0:03% caprylic acid in 0.075% PTC combined with 0:07% caprylic acid in 0.0875% SGM in 100 mM sodium citrate containing 0.5% Methocel.
  • microbicidal and adhesion inhibitory properties of all three test preparations were assessed using the standard viability and adhesion inhibition assay described in the Methods.
  • the inoculum for both assays was an 18 hour broth culture of Candida albicans , grown in YEPD medium and concentrated ⁇ 10 by centrifugation and re-suspension in one tenth volume of supernatant.
  • the fast acting PTC+Cap behaved as expected reducing viability to zero detectable cells in 20 minutes.
  • the slow acting SGM+Cap was also as expected with viability being reduced by 5 logs in 55 minutes.
  • the combination 30:70 blend gives a good example of a ‘tailored release’ profile, viability ,was reduced by more than 2 logs in 10 minutes, a rate which paralleled the fast acting PTC+Cap, thereafter the microbicidal rate slowed considerably, and approximated to that of the slow acting SGM+Cap.
  • the blend however had achieved 1 log greater reduction in viability at 55 minutes compared to SGM+Cap alone.
  • Washed yeast cell pellets were re-suspended in 2.5 ml volumes of the test preparations (PTC, PTC+Cap, SGM, SGM+Cap, 30:70 blend described above and BSA blank). 100 mM sodium citrate at pH 4.5 was used for determination of control adhesion. After 10 minutes pre-exposure, the yeast suspensions were used to re-suspend washed Buccal Epithelial Cell pellets which had been harvested and prepared as described in the Methods.
  • the combined yeast and Buccal Epithelial cell in test, blank or control were incubated with gentle agitation for 60 minutes at 37° C., after which direct microscopic counts using a Hemocytometer slide were used to enumerate the numbers of yeast adhering to 100 Buccal Epithelial cells: The results are presented in Table 16 and illustrated in FIG. 8 .
  • the relatively non-pathogenic Escherichia coli K12 NCTC 10538 was used (The National Collections of Industrial and Marine Bacteria Ltd, UK: Catalogue of Type Strains ISBN No 0 9510269 3 3).
  • the bacterium is routinely cultured and maintained on tryptone Soya agar or broth (TSB), which may be acquired commercially from Oxoid, UK and has the following constituents: 1.5% W/W tryptone (pancreatic digest of casein), 0.5% W/VV peptone (papaic digest of soybean meal), 0:6% WM/ sodium chloride; and agar at 1.5% VV/W when required as a solid medium.
  • TTB tryptone Soya agar or broth
  • the hands are sampled by dipping the tips of the fingers and thumb of each hand into 10 ml of sterile PBS in a Petri-dish, and rubbed against the base of the plate for 1 minute. After sampling, the hands are treated with either the product of this invention or Spirigel; 4 ml of either preparation is applied to the hands and manipulated over the surface area of both hands. The hands are then rinsed under clean (potable) running water, which is lukewarm at approximately 37° C. for a timed period of 20 seconds. After rinsing, the hands are held in an upright position while an assistant dries the palms and wrists with a paper towel. The finger tips and thumb are then sampled by immersion in 10 ml of PBS as described above.
  • a test preparation of the product of this invention was prepared, being a combination of caprylic acid in phosphatidyl choline at 30% and caprylic acid in sphingomyelin at 70% prepared as described in the Methods.
  • the membrane lipids were prepared as 1% concentrates with 1% caprylic acid and blended at 30:70 ratio.
  • a viscosity-enhancing agent is employed to improve the rheology of the test preparation.
  • a Carbopol co-polymer Pemulen TR-1 from Noveon Inc, Cleveland, Ohio was used at 0.45%.
  • the polymer was added to a volume of absolute ethanol equivalent to 70% of final preparation volume and allowed to disperse therein for 30 minutes.
  • a 30% volume of 30:70 blend of the product of this invention as described above was then added to the ethanol and polymer with constant stirring to facilitate rapid dispersion.
  • Spirigel is reported to contain 70% ethanol, 30% water and an unknown amount Of an unknown Viscosity-enhancing agent.
  • Brisket has an optimal distribution of lean muscle, fat and collagen, and is consequently considered to be representative of all potentially infected wound surfaces.
  • Brisket sections are excised immediately post slaughter, without chilling and with the external fascia membrane intact, these are divided under aseptic conditions into cubes of approximately 1 cm square. Prepared cubes are ‘infected’ by submerging them in late log phase cultures of bacteria for one minute, dried and suspended in a 37° C. environment for one hour to facilitate bacterial adherence and colonization of the meat surfaces.
  • a suitable example of a wound care formulation is the “standard formulation” of this invention (containing 0:5% caprylic acid in 0.4% DLL) diluted 1:1 in 200 mM sodium citrate buffer at pH 4.5, which then consists of 025% caprylic acid in 0.2% DLL in 100 mM sodium citrate buffer at pH4.5
  • Test sections of infected meat are treated by spraying the wound care formulation directly onto the infected surface and evaluating the test samples for residual bio-burden at predetermined exposure times infection and its eradication are confirmed by mechanical maceration of treated and untreated sections in sterile phosphate buffered saline (PBS) and enumeration of the bio-burden by standard microbiological serial dilution and plate counting techniques.
  • PBS sterile phosphate buffered saline
  • FIG. 9 Typical results are presented in FIG. 9 , where greater than 7 logs of the common wound infecting bacteria, Staphylococcus aureus and Streptococcus pyogenes are shown to be eradicated in less than 120 minutes. It should be appreciated that the rate of kill on a fissured surface such as a wound is extended due to the nature of the surface and the time required for the formulation to permeate to the seat of the infection.
  • FIG. 10 A comparison of the relative potency of the standard formulation in citrate buffer as above with the alternative and commonly used antimicrobials, chlorhexidine gluconate and silver sulphadiazine is presented in FIG. 10 .
  • the product of this invention has virtually eradicated 7 logs of Staphylococcus aureus: 1 log remains under chlorhexidine treatment and 3.5 logs with silver sulphadiazine.
  • Staphylococcus aureus RN4220 is noted for its ability to form tenacious biofilm under laboratory conditions when stress cultured in the presence of sodium chloride.
  • the organism was cultured to mid log phase (10 hours) in Brain Heart Infusion broth and 20 micro-liter volumes used to inoculate wells of a 96 well microtitre plate containing 180 ⁇ l of BHI supplemented with 4% sodium chloride. When incubated at 37° C. under these conditions for 6 hours an appreciable biofilm is formed at the base of each well.
  • the biofilm is quantified by decanting the culture and washing the wells with sterile distilled water, after which the plates are dried and stained with 0.4% crystal violet, re-washed and dried; the Optical Density of the stained biofilm is measured at 570 nm.
  • Alamar Blue is a redox indicator from Invitrogen Ltd, Paisley, UK. It imparts a deep blue color to the media, and when reduced by microbial metabolic activity the Color changes from non-fluorescent blue to a highly fluorescent red; absorbance and emerging fluorescence may be measured at 570 nm and 600 nm.
  • Microtitre plate wells containing pre-formed biofilm were treated with the membrane lipid CLS (ML CLS) used in Example 7 and similar wells were also treated with Zuragen, Duralock and Taurosept for time periods ranging from zero to 60 minutes. At the end of each exposure period, the plates were decanted and washed once with phosphate buffered saline containing 3% Tween 80 and twice with sterile distilled water. 200 ⁇ l of BHI containing 10% Alamar Blue was added to the wells and the plates incubated for 60 minutes. There was no detectable color change in any of the wells treated with the membrane lipid CLS of this invention indicating complete eradication of viability within the biofilm in less than one hour. All of the wells treated with Zuragen, Duralock or Taurosept had changed from blue to red demonstrating little or no reduction in viability in the established biofilm.
  • MCLS membrane lipid CLS
  • Microtitre plate went containing pre-formed biofilm were treated with the four formulations for time periods from zero to 60 minutes, decanted and washed as described previously.
  • the treated plates were dried and stained with 0.4% crystal violet, dried and the intensity of stain being a measure of residual biofilm was recorded at 570 nm.
  • the results are illustrated in FIG. 11 wherein it is evident that although some reduction in biofilm was achieved with Zuragen, Duralock and Taurosept, it is inconsequential in comparison to the near total eradication of biofilm achieved with the membrane lipid CLS (ML CLS) of this invention.
  • MCLS membrane lipid CLS
  • De-lipidised lecithin is a more potent adhesion inhibitory substance compared to milk serum apo-proteins in WO 03/018049, wherein the apo-proteins are lipase hydrolysis of whey proteins.
  • a whey protein hydrolysate was prepared As described in WO 03/018049 using Carbelac 80 whey protein concentrate.
  • a whey protein isolate (Provon 190 from Glanbia PLC) was also tested contemporaneously.
  • a formulation of 0.5% W/V caprylic acid in 0.4% W/V de-lipidised lecithin was prepared in 100 mM sodium citrate pH 4.5 as described in the Methods and included in the test sequence. All test items were dispersed in 100 mM sodium lactate buffer at pH 4.5. The results are presented in Table 18 below.
  • the MIC by agar dilution technique is also used here to demonstrate the superior potency of the “standard formulation” described hereinabove over the product disclosed in WO2009/072097 comprising a blend of free fatty acids emulsified in the whey protein isolate, Provan 190 from Glanbia PLC.
  • the product of WO2009/072097 contains 28% by weight of free fatty acid blend, while the standard formulation of this inventions contains just 0.5% by weight.
  • the product of WO2009/072097 was diluted by 5/28 in sterile distilled water to obtain a dispersion comprising 5.0% free fatty acid which was then diluted further and used to prepare agar plates ranging from 1.0% free fatty acid to 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05% and 0.025% (of fatty acid content).
  • the measured MIC for the standard formulation is one quarter or less than that of the product of WO2009/072097, indicating a four times greater potency.
  • the measured MIC is one half to one third less than the product of WO2009/072097 again clearly demonstrating significantly greater potency.
  • the measured MIC's for the emulsion of caprylic acid in Provon 190 are significantly greater (significantly less potent) than the standard formulation of this invention.
  • Fatty acids in free form have relatively little microbicidal effect primarily because of their insolubility in aqueous medium.
  • Emulsification in membrane lipids as described herein expands the relative surface area of the fatty acid and facilitates its dispersal in aqueous medium.
  • the membrane lipid emulsification agent also facilitates contact and transfer of the fatty acid to a microbial cell surface.
  • variable lipophylicity between different membrane lipids affects the rate of microbicidal effect.
  • membrane lipids are superior emulsification agents, exhibiting superior microbicidal effect as demonstrated in Example 15. As demonstrated here the superior potency of membrane lipid emulsified free fatty acid extends across a range of microbicidal fatty acids.
  • Emulsions of seven different free fatty acids at 0.5% W/V were prepared in 0.4% W/V de-lipidised lecithin as described, in the methods. Emulsions were prepared at temperatures above the melting points of the individual free fatty acids: caproic, caprylic and pelargonic at room temperature (20° C.); capric and undecylenic acid were prepared at 37° C., lauric acid was emulsified at 50° C. and myristic at 60° C. A blend of 50% caproic and 50% lauric acid has a melting point of less than 28° C. as does a blend of 49% leuric acid in oil of lemon balm, and these were emulsified at 37° C.
  • each individual fatty acid and blend thereof in free non-emulsified form and emulsified in de-lipidised lecithin was evaluated using the microbicidal suspension test described in the methods. Evaluation of the microbicidal effect of free non-emulsified fatty adds is frustrated by their insolubility. Inclusion of the non-emulsified form was considered necessary to illustrate the exponential increase in potency in the emulsified form.
  • Each free fatty acid was prepared at 0.5% W/V in water and agitated vigorously to disperse followed by immediate pipetting of 1.0 ml aliquots to test containers, prior to inoculation with the test organism. The test organism was Staphylococcus aureus RN 4220 grown to late log phase on Brain Heart Infusion Broth. The results are presented in Table 20 below.
  • Free fatty acids in non-emulsified form achieve at best 1 log reduction in viability over the test period of 6 minutes.
  • the membrane lipid emulsification agent (de-lipidised lecithin) equally exerts little or no microbicidal effect
  • equivalent weight emulsions of all other fatty acids and blends thereof in de-lipidised lecithin reduce residual viability in a test inoculum of 6.94 to 7.87 logs to zero in less than 4 minutes.
  • De-lipidised lecithin on its own in the test achieves 81% inhibition of adhesion.
  • Emulsions of caproic, caprylic, pelargonic, capric and undecylenic achieve greater than 99% inhibition, and emulsions of lauric and myristic achieve greater than 86% inhibition. All non-emulsified free fatty acids achieve less than 17% inhibition.
  • the adhesion inhibitory effect of caprylic acid for example is amplified by a factor of 14 in emulsified form.
  • the adhesion inhibitory effect is essentially swamped by the intrinsic microbicidal effect. It has been demonstrated that the majority of microbial cells will be dead as a result of exposure to the microbicidal effect of the emulsified fatty acid, and it must be assumed that this will influence adhesion.
  • a more measure of the amplified adhesion inhibitory effect attributable to emulsions vs free acid or non-emulsified membrane lipids may be obtained using a concentration below the Minimum inhibitory Concentration (MIC) of the emulsified free fatty acid.
  • the MIC of caprylic acid in the formulation of this invention is greater than 0.1%.
  • a formulation of 0.5% caprylic in 0.4% de-lipidised lecithin (as used above) is diluted by a factor of 5 in sterile distilled water to achieve a concentration of 0.1% caprylic in 0.08% de-lipidised lecithin and further diluted by half to achieve 0.05% caprylic in 0.04% DLL. These dilutions together with the same concentrations of DLL and non-emulsified free caprylic acid were tested in the Buccal Epithelial Cell assay as above. The results are presented in Table 22.
  • caprylic acid As illustrated in Table 22, the addition of caprylic acid at concentrations below its MIC (0.1% and 0:05%) amplifies the adhesion inhibitory effect of 0.08% DLL and 0.04% DLL by more than a factor of three in both cases.
  • Mammalian cell membranes are susceptible to disruption by free fatty acids in a manner not dissimilar to their effect on microbial cell membranes.
  • the effect is not classical cell toxicity as it relates to superficial cell surface damage and not interference with metabolic process or nucleic acid replication. It is ameliorated significantly by body fluids in vivo. Protection against mammalian cell membrane damage can be enhanced by adding additional amounts of free membrane lipid to a membrane lipid emulsion of a free fatty acid. The protective effect is not confined to the use of membrane lipids.
  • milk serum whey protein isolate also serves as a suitable, although not optimal barrier against mammalian cell damage.
  • the test item is an emulsion of 0.5% caprylic acid in 0.4% de-lipidised lecithin prepared as described in the Methods, and combined with an equal volume of 200 mM sodium citrate at pH 4.5 also prepared as described in the Methods.
  • Dispersions of 0.4% and 0.8% de-lipidised lecithin were prepared in 200 mM sodium citrate at pH 4.5, and combined in equal volumes with aliquots of the emulsion of caprylic acid in de-lipidised lecithin to achieve emulsions of 0.25% caprylic acid in 0.2% de-lipidised lecithin suspended in an aqueous solution of 100 mM sodium citrate at pH 4.5 in which further-amounts of either 0.2% or 0.4% de-lipidised lecithin were dispersed, these are described as Test+0.2% DLL or Test+0.4% DLL
  • the % cell survival in the test item is just 16% after 60′ minutes.
  • the control consisting of Raji B cells in cell culture media lost just 8% viability and the buffer blank being 100 mM sodium citrate at pH 4.5 was even less at 4% reduction.
  • the addition of 0.2% and 0.4% free membrane lipid (de-lipidised lecithin) % cell survival in the test was increased from 16% to 77% and 93%, and although not quite as effective, the addition of free WPI increased cell survival from 16% to 63% and 67%.
  • Bovine serum albumin was considerably less effective in protecting against cell damage.
  • a late log phase culture of the yeast Candida albicans grown as described in the Methods contained 1.33 ⁇ 10 7 viable cells per ml. This was used to inoculate aliquots of each test item from above at a dilution of 1:10 such that each ml of test item contained in excess of 6 logs of yeast cells. Samples were withdrawn over time periods of up to 10 minutes and assessed for residual viability using the procedures described in the Methods. As illustrated in Table 24 below, detectable viability was eradicated in less than 5 minutes by all test items.
  • Marvel skim milk powder from Premier International Foods (UK) Ltd, Spalding, Lincolnshire, England was re-constituted using 90% of the water volume according to the manufacturer's instructions. Once fully hydrated 10% by volume of the combined mix of separately emulsified free fatty acids was added and mixed by stirring, bringing the total volume to 100%.
  • Helicobacter pylori was grown on Columbia Blood Agar supplemented with 5% de-fibrinated sheep blood in an anaerobic jar using Anaerogen low oxygen gas packs from Oxoid UK. Salmonella typhimurium and E. coli K12 were grown on Brain Heart Infusion agar as described in the Methods.
  • microbicidal efficacy of the re-constituted milk supplemented with the three individually emulsified free fatty acids was determined using the Minimum Inhibitory Concentration (MIC) method of agar dilution described in the methods. Dilutions of the re-constituted skim milk were prepared in sterile distilled water such that further dilutions of aliquots of these in cooled agar provided an agar with combined free fatty acid concentrations ranging from 1% to 0.1% in 0.1% increments and from 0.1% to 0.01% in increments of 0.01%. Cultures of the three test organisms were inoculated onto these plates and incubated according to culture requirements: Helicobacter under low oxygen tension, Salmonella and E. coli under aerobic conditions all at 37° C.
  • MIC Minimum Inhibitory Concentration
  • the minimum Inhibitory Concentration, being the lowest dilution where no growth was observed was greater than 0.5% for Helicobacter and greater than 0.1% for both Salmonella and E. coli.

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WO2016115639A1 (en) * 2015-01-23 2016-07-28 Biocidium Biopharmaceuticals Inc. Anti-bacterial compositions
US9555116B2 (en) 2009-11-17 2017-01-31 Michael Anthony Folan Antimicrobial compositions containing free fatty acids
US10772343B2 (en) 2014-11-19 2020-09-15 Kansas State University Research Foundation Chemical mitigants in animal feed and feed ingredients
US11045589B2 (en) 2017-09-22 2021-06-29 Becton, Dickinson And Company 4% trisodium citrate solution for use as a catheter lock solution
US11065223B2 (en) 2016-02-22 2021-07-20 Board Of Regents, The University Of Texas System Antimicrobial compositions and uses thereof
US11266604B2 (en) 2015-09-17 2022-03-08 Alaya (Shanghai) Bioscience Co., Ltd. Highly stable non-vesicular nanoparticles and application thereof in treating microbial infection
US11298379B2 (en) * 2020-04-16 2022-04-12 James F. Kane Compositions comprising milk fat globules and methods of making same
US11452291B2 (en) 2007-05-14 2022-09-27 The Research Foundation for the State University Induction of a physiological dispersion response in bacterial cells in a biofilm
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US11452291B2 (en) 2007-05-14 2022-09-27 The Research Foundation for the State University Induction of a physiological dispersion response in bacterial cells in a biofilm
US9555116B2 (en) 2009-11-17 2017-01-31 Michael Anthony Folan Antimicrobial compositions containing free fatty acids
US11213503B2 (en) 2009-11-17 2022-01-04 Westgate Biomedical Ltd. Antimicrobial compositions containing free fatty acids
US10300176B2 (en) 2013-11-25 2019-05-28 Medline Industries, Inc. Catheter lock solution formulations
WO2015077798A1 (en) * 2013-11-25 2015-05-28 Excelsior Medical Corporation Catheter lock solution formulations
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US10772343B2 (en) 2014-11-19 2020-09-15 Kansas State University Research Foundation Chemical mitigants in animal feed and feed ingredients
US10918118B2 (en) 2014-11-19 2021-02-16 Kansas State University Research Foundation Chemical mitigants in animal feed and feed ingredients
US11896035B2 (en) 2014-11-19 2024-02-13 Kansas State University Research Foundation Chemical mitigants in animal feed and feed ingredients
US11363814B2 (en) 2015-01-23 2022-06-21 Biocidium Ip Holdco, Co. Anti-bacterial compositions
US10716305B2 (en) 2015-01-23 2020-07-21 Biocidium Biopharmaceuticals Inc. Anti-bacterial compositions
WO2016115639A1 (en) * 2015-01-23 2016-07-28 Biocidium Biopharmaceuticals Inc. Anti-bacterial compositions
JP2018509464A (ja) * 2015-01-23 2018-04-05 バイオシディウム バイオファーマスーティカルズ インコーポレイテッド 抗菌組成物
US11266604B2 (en) 2015-09-17 2022-03-08 Alaya (Shanghai) Bioscience Co., Ltd. Highly stable non-vesicular nanoparticles and application thereof in treating microbial infection
US11065223B2 (en) 2016-02-22 2021-07-20 Board Of Regents, The University Of Texas System Antimicrobial compositions and uses thereof
US11826332B2 (en) 2016-02-22 2023-11-28 Board Of Regents, The University Of Texas System Antimicrobial compositions and uses thereof
US11045589B2 (en) 2017-09-22 2021-06-29 Becton, Dickinson And Company 4% trisodium citrate solution for use as a catheter lock solution
US11298379B2 (en) * 2020-04-16 2022-04-12 James F. Kane Compositions comprising milk fat globules and methods of making same
KR20230108531A (ko) * 2022-01-11 2023-07-18 국민대학교산학협력단 베헨산을 포함하는 바이오필름 형성 억제용 조성물
KR102659787B1 (ko) 2022-01-11 2024-04-23 국민대학교산학협력단 베헨산을 포함하는 바이오필름 형성 억제용 조성물

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