Patent Description:
The oral cavity contains many different species of bacteria. Some species of oral pathogenic bacteria have been implicated in the development of periodontal disease and tooth decay however it is believed that certain species of oral bacteria are beneficial for maintaining oral health. Without being bound by any theory, it is believed that lysine degradation by oral bacteria contributes to gingival deterioration and that biosynthesis of lysine by the oral microbiome can positively contributes to the promotion of gum health.

Prebiotic oral care compositions are disclosed in <CIT>, <CIT>, <CIT> and <CIT>.

It is believed that selective stimulation of beneficial oral bacteria to produce lysine may provide a valid preventative approach for oral health, for example in the prevention of periodontitis and gingivitis.

There remains a need for a composition that stimulates beneficial oral bacteria to produce lysine and thus aid the prevention of periodontitis.

The present invention relates to an oral care composition as defined in the appended claims.

The invention further relates to an oral care composition, as defined in the appended claims, comprising asparagine or salt thereof for use in preventing one or more of periodontitis, gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, gingival deterioration and detachment.

All amounts are by weight of the composition, unless otherwise specified.

It should be noted that in specifying any ranges of values, any upper value can be associated with any particular lower value.

Any ingredients mentioned in this application that are natural or naturally derived have been sourced from Europe.

As discussed above the disclosure relates to an oral care composition comprising i) asparagine or salt thereof; and ii) anti-microbial agent.

The disclosure also relates to asparagine or salt thereof for use in the treatment of periodontitis, gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, gingival deterioration and detachment and mixtures thereof.

A further aspect of the disclosure is the use of compound asparagine or salt thereof in the manufacture of a medicament for the treatment of periodontitis, gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, gingival deterioration and detachment and mixtures thereof.

The asparagine or salt thereof can be of any chirality preferably it is L-asparagine.

Preferably the level of asparagine or salt thereof is from <NUM> to <NUM> wt% of the total composition, more preferably from <NUM> to <NUM> wt%. most preferably from <NUM> to <NUM> wt%.

The composition according to the disclosure comprises an anti -microbial agents, preferred anti-microbial agents are a source of fluoride in which the source of fluoride is preferably selected from the group consisting of sodium fluoride, stannous fluoride, sodium monofluorophosphate, zinc ammonium fluoride, tin ammonium fluoride, calcium fluoride, cobalt ammonium fluoride and mixtures thereof.

In a further aspect of the disclosure preferred anti-microbial agents are water-soluble or sparingly water-soluble sources of metal salts. Preferred are zinc ions such as zinc chloride, zinc acetate, zinc gluconate, zinc sulphate, zinc fluoride, zinc lactate, zinc oxide, zinc monoglycerolate, zinc tartrate, zinc pyrophosphate and zinc maleate; also preferred are stannous ions such as stannous fluoride and stannous chloride. Particularly preferred is zinc citrate.

Further suitable anti-microbial agents include ethyl lauroyl arginate HCl, chlorhexidine, sanguinarine extract, metronidazole, quaternary ammonium compounds, such as cetylpyridinium chloride; cetylpyridium chloride clay complex, bis-guanides, such as chlorhexidine digluconate, hexetidine, octenidine, alexidine; and halogenated bisphenolic compounds, such as <NUM>,<NUM>' methylenebis-(<NUM>-chloro-<NUM>-bromophenol).

Preferred levels of anti-microbial agents are from <NUM> to <NUM> wt% of the total composition, more preferably from <NUM> to <NUM> wt%, most preferably from <NUM> to <NUM> wt%.

It is preferable if the oral care toothpaste composition comprises fumaric acid or sodium fumarate Preferably the level of fumaric acid or salt thereof is from is from <NUM> to <NUM> wt% of the total composition, preferably from <NUM> to <NUM> wt%. most preferably from <NUM> to <NUM> Wt%.

In a preferred embodiment of the disclosure the weight ratio of asparagine to fumaric acid or salt thereof is from <NUM>:<NUM> to <NUM>:<NUM>.

The composition of the disclosure is used to clean the surfaces of the oral cavity and is known as an oral care composition.

Accordingly, product forms for compositions of the disclosure are those which are suitable for brushing and/or rinsing the surfaces of the oral cavity.

In one preferred embodiment of the disclosure the composition is in the form of a dentifrice. The term "dentifrice" denotes an oral composition which is used to clean the surfaces of the oral cavity. Such a composition is not intentionally swallowed for purposes of systemic administration of therapeutic agents, but is applied to the oral cavity, used to treat the oral cavity and then expectorated. Typically such a composition is used in conjunction with a cleaning implement such as a toothbrush, usually by applying it to the bristles of the toothbrush and then brushing the accessible surfaces of the oral cavity.

A composition according to the disclosure (such as a dentifrice/toothpaste) will generally contain further ingredients to enhance performance and/or consumer acceptability, in addition to the ingredients specified above.

Compositions according to the invention, toothpastes, preferably comprise particulate abrasive materials such as silicas, aluminas, calcium carbonates, dicalciumphosphates, calcium pyrophosphates, hydroxyapatites, trimetaphosphates, insoluble hexametaphosphates and so on, including agglomerated particulate abrasive materials, usually in amounts between <NUM> and <NUM>% by weight of the oral care composition.

Preferably the toothpaste, comprises a silica based abrasive. The preferred abrasive silicas used in the present invention is a silica with a low refractive index. It may be used as the sole abrasive silica, or in conjunction with a low level of other abrasive silicas, e.g. those according to <CIT>. The low refractive index silicas, used as abrasives in the present invention are preferably silicas with an apparent refractive index (R. ) in the range of <NUM> - <NUM>, preferably <NUM> - <NUM>, preferably having a weight mean particle size of between <NUM> and <NUM>, a BET (nitrogen) surface area of between <NUM> and <NUM><NUM>/g and an oil absorption of about <NUM> - <NUM><NUM>/<NUM>, but abrasive silicas with a lower apparent refractive index may also be used. Typical examples of suitable low refractive index abrasive silicas (e.g. having an R. of between <NUM> and <NUM>) are Tixosil <NUM> and <NUM> ex Rhone Poulenc; Sident <NUM> ex Degussa; Zeodent <NUM> ex Evoik Zeodent <NUM> ex Evonik, Sorbosil AC <NUM> ex PQ Corporation (having an R. of approximately <NUM>). The amount of these silicas in the composition generally ranges from <NUM>-<NUM>% by weight, usually <NUM>-<NUM>% by weight.

The toothpaste preferably comprises an inorganic or a natural or synthetic thickener or gelling agent in proportions of about <NUM> to about <NUM>% by weight depending on the material chosen. These proportions of thickeners in the dentifrice compositions of the present disclosure form an extrudable, shape-retaining product which can be squeezed from a tube onto a toothbrush and will not fall between the bristles of the brush but rather, will substantially maintain its shape thereon. Suitable thickeners or gelling agents useful in the practice of the present invention include inorganic thickening silicas such as amorphous silicas available from Huber Corporation under the trade designation Zeodent <NUM>, Irish moss, iota-carrageenan, gum tragacanth, and polyvinylpyrrolidone.

Compositions according to the invention preferably comprise a polymeric deposition aid. Preferably the composition comprises acid anhydride polymers, particularly preferred are co-polymers of maleic anhydride with methyl vinylether, in which the anhydride moiety may be in a partially or fully hydrolysed or alcoholysed form. Preferred copolymers include Gantrez(R) polymers such as:.

Particularly preferred co-polymers of maleic acid and methyl vinylether have a molecular weight of <NUM>,<NUM>,<NUM> or greater and an especially preferred material is Gantrez S-<NUM>.

Compositions according to the invention may comprise a tooth whitening agent. The whitening agent preferably comprises a green and/or a blue pigment. In the context of the present invention a pigment is generally understood to be a shade/material which is insoluble in the relevant medium, at the relevant temperature. This is in contrast to dyes which are soluble. In the context of this invention, the "relevant medium" is human saliva, the liquid medium in which the composition is used, at the temperature of the oral cavity during brushing of the teeth, i.e. up to <NUM> Degrees C. As a reasonable approximation, the relevant medium may be considered to be water and the relevant temperature to be <NUM> Degrees C.

Preferably the blue pigment is Pigment Blue <NUM>, more preferably Pigment Blue <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM> or <NUM>:<NUM>, most preferably <NUM>:<NUM>. A preferred pigment is blue pigment is Phthalocyanine Blue Pigment, Cl No. <NUM>, blue covarine.

The preferred Green pigment is Phthalocyanine Green, preferably Phthalocyanine Green CI-<NUM>.

Preferably the total level of pigment in the composition is from <NUM> wt% to <NUM> wt, more preferably from <NUM> to <NUM> wt%.

If the composition is a toothpaste it may be a dual phase paste, with the whitening pigments present in one phase.

Compositions according to the invention may comprise oral care enzyme systems such as hydrogen peroxide producing enzyme systems (e.g. the oxidoreductase enzyme glucose oxidase), amyloglucosidase, dextranase and/or mutanase, (optionally in the presence of zinc ion providing compounds and/or <NUM>- hydroxyquinoline derivatives), lactoperoxidase, lactoferrin, lysozyme and mixtures thereof.

In one embodiment a preferred class of oral care active for inclusion in the compositions of the invention includes agents for the remineralisation of teeth. The term "remineralisation" in the context of the present invention means the in situ generation of hydroxyapatite on teeth.

A specific example of a suitable agent for the remineralisation of teeth is a mixture of a calcium source and a phosphate source which, when delivered to the teeth results in the in situ generation of hydroxyapatite on teeth.

Illustrative examples of the types of calcium source that may be used in this context (hereinafter termed "remineralising calcium sources") include, for example, calcium phosphate, calcium gluconate, calcium oxide, calcium lactate, calcium glycerophosphate, calcium carbonate, calcium hydroxide, calcium sulphate, calcium carboxymethyl cellulose, calcium alginate, calcium salts of citric acid, calcium silicate and mixtures thereof. Preferably the remineralising calcium source is calcium silicate.

The amount of remineralising calcium source(s) (e.g. calcium silicate) in the composition of the invention typically ranges from <NUM> to <NUM>%, preferably from <NUM> to <NUM>% by total weight remineralising calcium source based on the total weight of the oral care composition.

Illustrative examples of the types of phosphate source that may be used in this context (hereinafter termed "remineralising phosphate sources") include, for example, monosodium dihydrogen phosphate, disodium hydrogen phosphate, sodium pyrophosphate, tetrasodium pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate, potassium dihydrogenphosphate, trisodium phosphate, tripotassium phosphate and mixtures thereof.

Preferably the remineralising phosphate source is a mixture of trisodium phosphate and sodium dihydrogen phosphate.

The amount of remineralising phosphate source(s) (e.g. trisodium phosphate and sodium dihydrogen phosphate) in the composition of this invention typically ranges from <NUM> to <NUM>%, preferably from <NUM> to <NUM>% by total weight remineralising phosphate source based on the total weight of the oral care composition.

Mixtures of any of the above described materials may also be used.

The composition according to the invention will comprise further ingredients which are common in the art, such as:.

The invention will now be illustrated by the following non-limiting Examples.

Neisseria elongata was grown overnight, the resulting growth was collected. The cultures were washed twice using phosphate buffered saline (PBS) and resultant pellet resuspended in 10mls of PBS for use in the experiment.

Each lysine generation experiment consisted of <NUM> CGVIII media, <NUM> <NUM>% glucose and <NUM> of either water, <NUM> fumarate or asparagine or combinations at <NUM> parts of each of the individual solutions of sodium fumarate, and asparagine. All solutions were adjusted to pH7 using sodium hydroxide. A <NUM> aliquot of bacterial culture was added to the mixed solutions (table <NUM>) Each combination was carried out in triplicate using <NUM> well plates.

A <NUM> sample was collected for baseline lysine measurement before placing the solutions on to the orbital shaker for incubation at <NUM> at 100rpm for <NUM> hours. A second <NUM> sample was collected after <NUM> hours for lysine measurement. Solutions without bacteria were also generated as controls for background assessment.

After collection the sample was centrifuged at <NUM> for <NUM> minutes at <NUM>,<NUM>, the supernatant was removed and frozen ready for analysis. Prior to analysis the samples were defrosted.

Lysine present in the solutions was measured using a fluorogenic kit supplied by Abcam (ab273311) following the manufactures instructions. Modifications were made to the assay with the addition of a lysine standard curve rather than spiking with a lysine standard. Aliquots of 10ul from each sample, standard and background solutions were added to individual wells of a black microtitre plate followed by the addition of 50ul of lysine assay buffer. The reagents of the kit were added together to form the analysis reagent and 40ul added to each sample, standard and background solution. The plate was incubated at <NUM> in the dark for <NUM> minutes prior to reading on a fluorescent plate reader at Ex/Em = <NUM>/<NUM>. The data was exported into excel, where a calibration curve was generated, and the absorbance of the sample minus the background solutions were compared to give lysine concentrations.

Concentrations of lysine were determined for each test solution and the mean levels of lysine for the replicate solutions generated Table <NUM> shows the levels of lysine generated by fumarate and asparagine, alone and in combination (mean and standard deviation). Levels of lysine above that of the media only control were found for all solutions with boosted levels for the combination. Statistical analysis was carried out by comparing the means using T-Test.

The results in Table <NUM> demonstrate that Neisseria elongata is capable of generating increased levels of lysine using asparagine.

Neisseria elongata was grown overnight in brain heart infusion (BHI) broth at <NUM> on an orbital shaker at 100rpm. The resulting growth was collected by centrifuging 2x30ml at 10000rpm for <NUM> minutes in <NUM> tubes. The cultures were washed twice using phosphate buffered saline (PBS) and resultant pellet resuspended in 10mls of PBS for use in the experiment.

Each lysine generation experiment consisted of <NUM> CGVIII media (see appendix), <NUM> <NUM>% glucose and <NUM> either water, <NUM> fumarate, aspartate or asparagine or combinations at <NUM> parts of each of the individual solutions, fumarate and aspartate of fumarate and asparagine. All solutions were adjusted to pH7 using sodium hydroxide and used to make a dilute toothpaste formulation using <NUM> of toothpaste plus <NUM> of each test solution. A <NUM> aliquot of bacterial culture was added to the mixed solutions (table <NUM>). Each combination was carried out in triplicate using <NUM> well plates.

Solutions were placed in an orbital shaker for incubation at <NUM> and 100rpm for <NUM> hours. A <NUM> sample was collected after <NUM> hours for lysine measurement. Solutions without bacteria were also generated as controls for background assessment.

After collection the samples was centrifuged at <NUM> for <NUM> minutes at <NUM>,<NUM>, the supernatant was removed and frozen ready for analysis. Prior to analysis the samples were defrosted.

Concentrations of lysine were determined for each test solution and the mean levels of lysine for the replicate solutions generated.

Claim 1:
An oral care toothpaste composition comprising:
i) a particulate abrasive
ii) asparagine or salt thereof;
iii) fumaric acid or sodium salt thereof.and
iv) an anti-microbial agent selected from the group consisting of a source of fluoride, a metal salt, ethyl lauroyl arginate HCl, chlorhexidine, sanguinarine extract, metronidazole, quaternary ammonium compounds, bis-guanides, halogenated bisphenolic compounds and mixtures thereof.