COMPOSITIONS CONTAINING NISIN

Abstract: The invention provides the use of a nisin composition for the manufacture of an oral hygiene composition for the control of candida characterised in that the composition comprises at least two components selected from a humectant, a metal ion chelator and a flavour, excluding any other antimicrobial agent, plus an orally acceptable carrier or excipient.

Description: COMPOSITIONS CONTAINING NISIN This invention relates to the use of oral hygiene compositions and in particular to stable mouthwashes and dentifrices having improved activity against orally important pathogenic microorganisms, including the fungi Candida. Candida which is present in the oral cavity may give rise to certain disease states, which may include thrush, and requires therapy. The control of Candida has now become an important part of general oral hygiene. More recently, it has been suggested that the polypeptide antibiotic nisin (Merck Index, 11th edn., entry 6481) may be of use in oral hygiene. Nisin is a lanthocin, containing the atypical amino acid lanthionine, produced naturally by various strains of the bacterium Streptococcus lactis. It is also a naturally occurring preservative found in low concentration in milk and cheese. Nisin has recently been recognised by the FDA as a direct food ingredient. A summary of the properties of nisin is to be found in Advances in Applied Microbiology 27 (1981), 85-123. A purified form of nisin has recently been made available by Applied Microbiology Inc., under the trade name Ambicin N. Oral care applications of nisin are disclosed in published application WO 93/11738 (SmithKline Beecham pic) and WO 89/12399 (Public Health Research Institute). The former discloses a dentifrice formulations whilst the latter discloses disinfectant compositions which may be in the form of oral rinses and in which nisin is combined with a non-bactericidal agent such as a surfactant or a chelating agent. These compositions however are only suitable for the protection against either Gram negative alone or in the latter case against Gram negative and positive bacteria. Nisin however has been shown to have limited activity against Candida species. The formulation of compositions comprising highly purified proteins, such as the compound nisin, as mentioned above are particularly challenging as a protein in this state is rendered sensitive to many of the processes that are commonly required in the preparation of successful formulations. These problems are highlighted in 'Stability of Protein pharmaceuticals' part B, In vivo Pathways of Degradation and Strategies for Protein Stabilization, Edited by Tim J. Ahern and Mark C. Manning. PCT published application no. WO 94/13143 (Applied Microbiology Inc.) discloses compositions containing a lanthionine bacteriocin such as nisin which are stabilised by the presence of a thioether stabilizing agent against degradation. Table 1 of WO/94 13143 shows a full nisin formulation, used in determining which component(s) was associated with the degredation of nisin. Results show that degradation can be minimised in formulations where the humectant glycerol is omitted. Further studies showed that nisin degradation was also dependent on the simultaneous presence of both EDTA and glycerol in the formulation. However neither compound caused an additional problem in the absence of the other. PCT published application no. WO 94/12105 (SmithKline Beecham pic) discloses compositions that comprise nisin plus an additional antimicrobial agent that are effective against orally important pathogenic microorganisms, including Candida, thus extending the antibacterial activity of nisin. Certain of the examples shown in 94/12105 disclose antibacterially effective compositions containing inter alia nisin, a further antimicrobial agent, humectant, chelating agent and flavour components. A simple solution of nisin alone did not show anticandida activity. We have effectively overcome the stability problems associated with purified proteins such as nisin and have surprisingly found that nisin in the presence of certain ingredients within a formulation has anticandida activity without the need for any other antimicrobial agent. Accordingly, the present invention provides the use of a nisin composition for the manufacture of an oral hygiene composition for the control of Candida characterised in that the composition comprises at least two components selected from a humectant, a metal ion chelator and a flavour, excluding any other antimicrobial agent, plus an orally acceptable carrier or excipient. Suitable humectants for use in compositions of the invention include for instance, glycerine, sorbitol, propylene glycol, polyethylene glycol and xylitol or mixtures thereof. Suitably the metal ion chelator is an orally acceptable salt of for example ethylenediaminetetraacetic acid (EDTA), or citric acid or an alkali metal salt thereof, for instance disodium hydrogen citrate, in accordance with the disclosure of WO 89/12399 (Public Health Research Institute of the City of New York). Preferably the metal ion chelator is disodium EDTA. Suitable flavours for use in the composition are those which may suitably include blends of mint and parts thereof with or without other essential oils. Examples of such mints include peppermint (Pipenta larvensis) and spearmint including various blends, and their constituents for example L- Carvone, Menthone and L-Menthol. Other suitable oils such as aniseed oil, eucalyptus oil, oil of wintergreen and thymol may also be employed. The level of flavour used will be dependant upon the ingredient selected, the sensory profile of the blend together with the perceived end use of the product. Suitably the flavour is present in amounts from 0.005-2.0% , preferably 0.05-1.5 % , more preferably 0.1-1.0% by weight of the composition. Accordingly, a preferred embodiment of the present invention provides for the use of an oral hygiene composition comprising nisin, glycerine, disodium EDTA and a flavour, excluding any other antimicrobial agent plus an orally acceptable carrier or excipient. Typically the humectant may be present in the range from 3 to 70%, preferably 15 to 30%, more preferably 5 to 30% by weight of the composition. Typically, the EDTA will be present in the range 0.005 to 10%, preferably 0.005 to 5% , more preferably 0.005 to 2.8% by weight of the oral hygiene composition. Suitably, in compositions of the present invention, nisin is used in a purified form, for instance the product sold under the trade name Ambicin N by Applied Microbiology Inc., 170 53rd Street, Brooklyn, New York, NY 11232, USA. Suitably, the oral hygiene composition comprises from 0.001 to 5.0%, preferably from 0.005 to 2.0% , advantageously from 0.01 to 1.0 % of nisin, by weight of the composition. In an alternative manner, the level of nisin needed is one which reaches a suffϊcent level in the oral cavity to inhibit the desired microrganisms. Oral hygiene compositions of the present invention may be presented in any of the formulations conventionally used in the art; for instance, as a mouthwash, dentifrice, including toothpaste and toothpowder, liquid toothpaste, gel or tablet. In a preferred aspect, the present invention is a mouthwash formulation. For a mouthwash preparation suitably nisin is present in a concentration of from 0.1 to 300μg/ml and the metal ion chelating agent present in a concentration of from 0.1 to 20mM. The components for the orally acceptable carrier or excipient will be selected according to principles well known to those skilled in the art for the preparation of such formulation types. Such components include a buffer etc.. thickening agent and abrasive, as appropriate, as well as other optional extras normally included in an oral care composition. Such components should be compatible with nisin. Thus, we have previously found (WO 93/11738, SmithKline Beecham) that polypeptide antibiotics such as nisin, gramicidin and tyrothricin are incompatible with anionic surfactants such as sodium lauryl sulphate and sodium N- methyl-N-cocyl laurate which are conventionally used in oral care compositions. Such anionic surfactants should preferably be avoided, in favour of nonionic, cationic or amphoteric surfactants. Suitable mouthwash formulations will have an aqueous base comprising water or aqueous ethanol in an amount of from 0-15%, preferably 5-12% by weight of the composition. It has been found that if level of alcohol is above 15 % then the nisin will begin to precipitate out of the composition. Mouthwash compositions may be provided in a "ready to use" form; as a concentrated solution, for dilution by the user immediately prior to use or in a non- alcohlol form; or in solid form, such as a tablet or in a sachet, for dissolution by the user immediately prior to use. Tablets may suitably be prepared using xylitol and/or sorbitol as the major ingredient. The sachets and tablets may be formulated to provide, on dissolution, a still mouthwash, or, by the incorporation of a suitable effervescent couple, for instance sodium carbonate/bicarbonatre and citric acid, an effervescent mouthwash. Suitably the composition further comprises a surfactant to solubilsise the flavour and/or to improve the sensory properties of the composition. . In a preferred aspect, a mouthwash composition according to the present invention comprises a surfactant or a combination of surfactants essential to solubilise the flavour system. In such a system where nisin itself has some surfactant properties and tends to absorb at the oil/ water interface, a solubiliser with a high HLB (hydrophilic-lipophilic balance) value is essential to impart stability. Suitably this implies surfactants with HLB values above 16, preferably above 20. This is especially noticeable when the formulation contains an alcohol component. Suitable nonionic surfactants include, for example, polyethoxylated sorbitol esters, in particular polyethoxylated sorbitol monoesters, for instance, PEG(40) sorbitan diisostearate, and the products marketed under the trade name 'Tween' by ICI; polycondensates of ethylene oxide and propylene oxide (poloxamers), for instance the products marketed under the trade name 'Pluronic' by BASF-Wyandotte and ' Synperonics' by ICI; condensates of propylene glycol; ■ polyethoxylated hydrogenated castor oil, for instance, the products marketed under the trade name 'Cremophors' by BASF-Wyandotte; and sorbitan fatty esters. Particularly preferred are the 'Synperonics' which may be represented by the following general formula: CH 3 HO-(OCH 2 CH2)a-(OCH-CH2)b-(OCH2CH2) a -OH Suitable examples include Synperonic PE/F68, F108 and F127. The most preferred is Synperonic PE/F108 which has an approximate molecular weight of 14,000 and an HLB (hydrophobic/lipophobic balance) value of 27. Also preferred are the polyethoxylated castor oils, examples of which include Cremophor RH40 and RH60. The most preferred of these being RH40 which has an HLB between 14 and 16. Suitable amphoteric surfactants include, for example, long chain imidazoline derivatives such as the product marketed under the trade name 'Miranol C2M' by Miranol; long chain alkyl betaines, such as the product marketed under the tradename 'Empigen BB' by Albright + Wilson, and long chain alkyl amidoalkyl betaines, such as cocamidopropylbetaine, and mixtures thereof. Advantageously, the surfactant is present in the range 0.005 to 20%, preferably 0.1 to 10%, more preferably 0.1 to 5% by weight of the composition. In the compositions of the present invention, it has been found that many of the more commonly used antioxidant materials are ineffective and a preferred stabiliser is the thoiether compound of formula (I) Rl - S - R2 (I) wherein R 1 is an alkyl group containing 1-6 carbon atoms or -(CH2)n-CH-COOH NH 2 and R 2 is - (CH 2 ) n -CH-R 4 R 3 wherein n is 0 to 5; R 3 is hydrogen or an amino group, or an hydroxyl group; and R 4 is a hydrogen, a carboxyl group, an ester group or an amido group wherein the amino function is contributed by an amino acid residue. or where in Rl and R2 are joined to form, with the sulphur, a thiazoline ring. In a preferred embodiment , the compound of formula (I) is the amino acid methionine or an analog thereof, examples of which include D methionine, DL methionine, methionyl glycine, N-acetyl DL methionine and N-acetyl D- methionine. The preferred analogue being the acetylated form of methionine, the D form being preferred over the racemate for taste reasons. Chemical thioether entities may also be used however some are not suitable owing to unacceptable odour or taste. A preferred chemical derivative is thiodipropionic acid. Suitably, the antioxidant is present in the composition in an amount of from 0.01% to 0.3%, preferably 0.03% to 0.25% by weight of the composition. In order to protect the composition from degredation, an oil soluble antioxidant may also included. Suitable antioxidants include the ' Tocopherols' , butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ascorbyl palmitate. Suitably the oil soluble antioxidant is present in amounts of from 0.005% to 0.05%, preferably 0.005% to 0.01 % by weight of the composition. Suitable abrasives for use in dentifrice compositions of the present invention include calcium carbonate, calcium phosphates, calcium pyrophosphate, insoluble sodium metaphosphate, sodium aluminosilicate, alumina, hydrated alumina, zinc orthophosphate, plastic particles, and silica, of which silica is the preferred abrasive. Suitable silicas include natural amorphous silicas, such as, for instance, diatomaceous earth, and synthetic amorphous silicas, such as precipitated silicas and silica gels, including silica xerogels. Suitable silica xerogels are described in US 3,538,230. Suitable grades of precipitated silicas have BET surface areas in the range 20 to 300, preferably 20 to 100 m^/g and median agglomerate sizes in the range 2 to 50, preferably 5 to 30μ. Suitable precipitated silicas and silica xerogels are those marketed under the trade names Sident and Syloblanc, by Degussa and W R Grace Corporation Davison Chemical Division, respectively. Advantageously, the silica is a "low anion" silica. As used herein, the term "low-anion" silicas refers to those in which anionic impurities such as sodium sulphate and sodium silicate which normally arise during the course of the manufacturing process are kept to a minimum, through careful control of the manufacturing process. "Low anion" silicas suitably have less than 1 %, preferably less than 0.5% advantageously less than 0.25% by weight of anionic impurities. Suitable such "low anion" silicas are described in EP 0 368 130 (Proctor & Gamble), EP 0 315 503 and EP 0 396 459 (Rhone-Poulenc) and WO 90/05113 (J.M. Huber Corp). Suitable "low anion" silicas include the grade RP93 available from Rhone-Poulenc. Suitably, compositions will have from 5 to 80%, preferably from 10 to 60% by weight of the abrasive. Compositions according to the present invention may usefully comprise a fluoride ion source, to provide an anti-caries activity. A fluoride ion source is found to be compatible with nisin. The appropriate fluoride source for the combination of nisin and antimicrobial agent will depend upon the particular antimicrobial agent chosen. The compatabilities, and incompatibilities, are well known and documented in the art for each of the suitable antimicrobial agent. Where applicable, therefore, suitable fluoride ion sources include metal fluoride salts, for instance alkali metal fluoride salts such as sodium fluoride, stannous fluoride, amine fluoride salts, alkali metal monofluorophosphate salts such as sodium monofluorophosphate and amine monofluorophosphate salts. Suitably the fluoride ion source would, if present, be included to provide from 50 to 3500 ppm, preferably 100 to 2500 ppm of fluoride ions. In addition to a humectant, compositions of the present invention may also contain further liquid such as, for instance, water, preferably deionised water. The orally acceptable vehicle or carrier may also comprise further optional ingredients such as sweetening agents, for example sodium saccharin, acesulphame K and aspartame. Sodium saccharin is preferred in amounts of from 0.005% to 0.01 % by weight of the composition. Dyes may also be present, of which the water soluble dyes are preferable, for example FD&C Yellow no.6 and patent blueV; whitening agents, for example titanium dioxide, preservatives, antisensitivity agents, such as strontium and potassium salts, and anticalculus agents, such as tetraalkali- and dialkali-imetal pyrophosphate salts. It will be appreciated that in each instance, an optional ingredient, if included, will be compatible with nisin and the antimicrobial agent. It is further appreciated that the combination of additional ingredients such as strontium will be in a manner compatible with other ingredients such as a fluoride ion source. Compositions according to the invention will have a pH which is orally acceptable and within which the antibacterial activity of nisin is not substantially compromised. Suitably, the pH is in the range 3.0 to 9.5, preferably in the range 3.5 to 6.5, more preferably between 4 and 5.5 . The compositions should be lightly buffered to ensure that pH in the mouth is above 4.0, preferably 5.0. Suitable buffer ingredients include alkali metal salts, preferably sodium salts such as sodium phosphate, sodium benzoate, sodium citrate, sodium acetate and sodium lactate. Preferably the buffer is the acetate or lactate, typically present in amounts from 0.005% to 1.0% by weight of the composition. Compositions according to the invention may be prepared by conventional processes comprising admixing the ingredients together in the appropriate relative amounts and finally, and if necessary, adjusting the pH to the desired value. Compositions of the present invention are intended for use in the prophyllaxis and/or treatment of common oral conditions and infections. Such infections include plaque outgrowth, gingivitis, periodontitis, lack of breath freshness, and Candida infections which may all be found within the oral cavity. These compositions may also be useful in the management of mouth ulcers. In particular, compositions of the present invention are effective against Candida related infections. Accordingly, in a further aspect, the present invention also provides a method of reducing or preventing the outgrowth of Candida, which method comprises applying an anticandida effective amount of a composition according to the present invention to a patient in need thereof. Accordingly, the present invention also provides for a method of treating or prophyllaxis of dental plaque. The invention will now be illustrated by reference to the following examples. Example 1 - Alcoholic Mouthwash W/W% Ambicin 0.03 Glycerin 12.57 Flavour 0.085 Ethanol (96%) 12.03 Disodium EDTA 0.037 Detergent 0.60 Sodium Saccharin 0.005 FD&C Blue no.1 0.0002 DL methionine 0.0298 Deionised water qs Example 2 - Alcoholic Mouthwash W/W% Ambicin 0.03 Glycerin 5.00 Flavour 0.12 Ethanol (96%) 12.00 Disodium EDTA 0.037 Sodium Saccharin 0.005 Patent Blue V 0.0002 DL methionine 0.0298 Sodium fluoride 0.05 Deionised water qs Example 3 - Alcoholic Mouthwash W/W% Ambicin 0.03 Glycerin 5.00 Flavour 0.12 Ethanol (96%) 12.00 Disodium EDTA 0.037 Sodium Saccharin 0.005 Patent Blue V 0.0002 N-acetyl D-methionine 0.24 Sodium fluoride 0.02 Detergent 0.30 Deionised water qs Example 4 - Non -alcoholic Mouthwash W/W Ambicin 0.03 Glycerin 5.00 Flavour 0.12 Disodium EDTA 0.037 Sodium Saccharin 0.005 Patent Blue V 0.0002 N-acetyl D-methionine 0.24 Sodium fluoride 0.02 Detergent 1.60 Deionised water qs

Claims: CLAIMS 1. Use of a nisin composition for the manufacture of an oral hygiene composition for the control of Candida characterised in that the composition comprises at least two components selected from a humectant, a metal ion chelator and a flavour, excluding any other antimicrobial agent, plus an orally acceptable carrier or excipient. 2. Use according to claim 1, wherein the humectant is selected from glycerine, sorbitol, propylene glycol and xylitol or mixtures thereof. 3. Use according to claim 1 or 2, wherein the metal ion chelator is an orally acceptable salt of ethylene diamine acetic acid (EDTA), or citric acid or an alkali metal salt. 4. Use according to claim 3, wherein the flavour is a blend of mint and parts thereof with or without other essential oils. 5. Use according to claim 4, wherein the blend of mint includes peppermint and spearmint. 6. Use according to any one of claims 1 to 5, wherein the humectant is present in the range from 3 to 7% by weight of the composition. 7. Use according to any one of claims 1 to 6, wherein the metal ion chelator is present in the range 0.005 to 10% by weight of the composition. 8. Use according to claim 7, wherein the composition comprises nisin, glycerine, disodium EDTA and a flavour, excluding any other antimicrobial agent, plus an orally acceptable carrier or excipient. 9. Use according to claim 8, wherein the composition is in the form of a mouthwash. 10. A process for the preparation of a composition according to any one of the above claims, which process comprises admixing the ingredients together in the appropriate relative amounts and finally, and if necessary, adjusting the pH to the desired value.

Inventors: BARTLETT MIKE, MCCONVILLE PETER SCOTT, PRICE FIONA, BARTLETT, MIKE, MCCONVILLE, PETER, SCOTT, PRICE, FIONA
Applicants: BARTLETT MIKE, MCCONVILLE PETER SCOTT, PRICE FIONA, SMITHKLINE BEECHAM PLC, BARTLETT, MIKE, MCCONVILLE, PETER, SCOTT, PRICE, FIONA
Application year: 1996
Main classification: A61K7
IPC codes: A61K8, A61Q11