Patent Description:
The main constituent of dental enamel is the basic mineral calcium hydroxyapatite (HAP), Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM>, which is inherently susceptible to the etching and dissolving action of acids. Examples of tooth decay that are caused by acids are dental caries and dental erosion. In dental caries, acidic end products of anaerobic bacterial metabolism in the dental plaque cause local dissolution of dental enamel, typically at sites which are difficultly accessible for dental hygiene. Dental erosion is the chemical dissolution of dental surfaces by acids from dietary or gastric origin, which, often in combination with mechanical wear (attrition and abrasion), can cause a widespread loss of superficial dental tissues. Under normal conditions saliva protects partially the enamel against the detrimental effects of acidic attacks by the neutralizing action of its buffer systems and by depositing a tooth pellicle, which is a lubricative film of salivary (glyco)proteins that covers the dental surfaces. Current consumption behavior has mounted up the acidic attacks to a level that surpasses the protective capacity of saliva. Worldwide this has led to a burgeoning incidence of dental erosion. Dental enamel is also susceptible to tartar formation (dental calculus). Dental calculus refers to a build-up of hardened (mineralized) plaque on the teeth, formed by the presence of saliva, debris, and minerals. Dental calculus is a deposit of calcium phosphate salts on the surface of the teeth. It comprises a mixture of calcium phosphate minerals such as brushite, octacaclium phosphate, tricalciumphosphate and biological apatite.

Conventional oral care compositions, such as toothpastes and mouthwashes, are particularly suited for prevention of caries and tartar formation. However, no formulation has yet successfully added significant protection against dental erosion. Therefore, new formulations to protect dental surfaces are urgently needed.

The disclosure provides the use of a sphingosine compounds having formula I, for the non-therapeutic cosmetic prevention of discoloration of a tooth.

Preferably, the use of the compounds and the resulting coatings prevent or reduce tooth demineralization, a tooth demineralization disorder (preferably selected from dental erosion, dental caries, and dentine hypersensitivity), gum disease, and/or the formation of dental calculus. Preferably, said compounds are used to protect the tooth from the acid erosion associated with a tooth demineralization disorder. More preferably, the compounds are used to prevent or reduce tooth demineralization or a tooth demineralization disorder (preferably selected from dental erosion, dental caries, and dentine hypersensitivity).

In one aspect, the disclosure provides a method for coating a hydroxyapatite surface or a hydroxyapatite containing material comprising contacting said surface or said material with a sphingosine compound having formula I. Preferably, the hydroxyapatite surface or hydroxyapatite containing material is bone or tooth or an artificial or prosthetic bone or tooth. Preferably, the method reduces or prevents acid erosion of the hydroxyapatite or reduces or prevents the build-up of salt precipitates on the surface or material.

In one aspect, the disclosure provides cosmetic treatments for preventing the discoloration of a tooth comprising providing a sphingosine compound having formula I to said tooth.

In one aspect, the methods, uses, and treatments described herein further comprise the provision of hydroxyapatite nanoparticles.

The disclosure provides compositions comprising a sphingosine compound having formula I. Preferably, the composition is an oral care composition, preferably selected from dentifrice (such as tooth powder and toothpaste), chewing gum, artificial saliva, and mouthwash. Preferably, the composition is a food composition, preferably selected from include dairy products, processed food products, oils, food and/or vitamin supplements, snack products, and beverage products. Preferably the compositions further comprise hydroxyapatite nanoparticles.

In one aspect, the disclosure provides a prosthetic bone or tooth coated with a sphingosine compound having formula I. Preferably the bone of tooth is also coated with hydroxyapatite nanoparticles.

The sphingosine compound having formula I is:
<CHM>.

More preferably, X is an alkyl or alkenyl having <NUM>-<NUM> carbons, preferably <NUM> to <NUM> carbons, more preferably a linear alkyl;.

Preferably, X is an alkyl or alkenyl having <NUM>-<NUM> carbons, preferably <NUM> to <NUM> carbons,R<NUM> is H;.

Preferably, X is an alkyl or alkenyl having <NUM>-<NUM> carbons, preferably <NUM> to <NUM> carbons,.

The present disclosure demonstrates the protective effect of surfaces/materials by lipid-containing compounds based on the family of sphingosines. Treatment of hydroxyapatite, the acid-sensitive mineral phase of dental enamel, with a sphingosine compound provided more than <NUM>% protection against dissolution by citric acid relative to untreated samples (see, e.g., <FIG> and <FIG>). Accordingly, methods are provided for protecting hydroxyapatite containing materials using a sphingosine compound. While not wishing to be bound by theory, it is believed that the sphingosine compounds adhere to hydroxyapatite forming a protective barrier. Said adherence renders these compounds useful in the protection against, e.g., acid erosion, biofilm formation, and tooth demineralization.

Preferably, the sphingosine compounds useful in the present disclosure comprise formula I:
<CHM>.

Preferably, the sphingosine compound is a phytosphingosine-based compound. Such compounds include N-tetracosanoyl phytosphingosine, N-stearoyl phytosphingosine, N-oleoyl phytsosphingosine, N-linoleoyl-phytosphingosine, N-(<NUM>-hydroxytetracosanoy-l), phytosphingosine, N-(<NUM>-hydroxyoctdecanoyl) phytosphingosine, N-phytosphingosine, <NUM>(2hydroxyoctdecanoyl) hydroxyoctdecanoyl) phytosphingosine, N-(<NUM>-stearoyloxy- hepatoaconsanoyl) phtosphingosine, N_(<NUM>-oleoyloxheptacosanoyl)phytosphingosine, N (<NUM>-linoleoyoxyheptaconsa- noyl) phytosphingosine, N-(<NUM>-stearoyloxytricosanoyl) phytosphingosine, N-acetyl-phytosphingosine, N-hexadecanoyl- phytosphingosine, N-hexanoyl- phytosphingosine, N-octadecanoyl- phytosphingosine, and N-octanoyl- phytosphingosine maybe used. In preferred embodiments, the sphingosine compound is a sphingolipid comprising a sphingoid base as described herein. The most preferred sphingosine compound is phytosphingosine. Also preferred compounds are phytosphingosine-phosphate and D-erythro-sphingosines, such as D-erythro-sphingosine C15.

Additionally, the present disclosure demonstrates that sphingosine compounds can prevent bacterial adherence to a surface. This makes them useful as agents to reduce or prevent biofilm formation. Such compounds can be used, e.g., as coatings on medical devices and surgical equipment. This effect also increases their usefulness in oral care and food compositions. The effect of the sphingosine compounds preventing bacterial adherence is separable from any effect as a bactericide. <CIT> describes compositions comprising PHS for the prevention of the formation of plaque and tartar in animals and for the treatment of odontostomatological pathologies of animals. In the methods and compositions (e.g., oral care and food compositions) described herein, the sphingosine compounds are not used as antimicrobials but rather to prevent bacterial adherence. Preferably, for these applications and compositions the sphingosine compound is selected from one or more of the following: PHS, PHS phosphate, stearoyl PHS, sphinganine, and sphingosine. Most preferred for the prevention of bacterial adherence is sphinganine. Preferably, more than one sphingosine compound is used in the methods and compositions as described herein. Preferably, PHS and sphinganine are used together in a method, composition, or coated on an article as described herein. Preferably the adherence of oral bacteria is reduced. Preferably the adherence of S. gordonii is reduced. Preferably the adherence of S. sanguinis is reduced. More preferably, the adherence of S. mutans is reduced.

The sphingosine compound may also be a conjugate of said compound, such as a sphingolipid. Sphingolipids comprise a complex range of lipids in which fatty acids are linked via amide bonds to a long-chain base or sphingoid. More precisely, sphingolipids consist of long-chain bases, linked by an amide bond to a fatty acid and via the terminal hydroxyl group to complex carbohydrate or phosphorus-containing moieties. Sphingoid bases include dihydrosphingosine (sphinganine), sphingosine, and phytosphingosine. Ceramides are a specific group of sphingolipids containing sphingosine, phytosphingosine or dihydrosphingosine as a base in amide linkage with a fatty acid. Sphingolipids suitable for the present invention have a sphingoid base having the formula of formula I as disclosed herein.

Suitable conjugates of sphingosine compounds also comprise the compounds of formula I conjugated to a protein or peptide moiety. Said moiety may improve the production, delivery, HAP targeting, stability, or efficacy of the sphingosine compound. Preferred peptide moieties are HAP binding moieties. A number of such peptides are known in the art including statherin, salivary agglutinin, polyglutamate, and casein peptides. Preferably, said peptide comprises the sequence DSpSpEEK (from statherin, wherein Sp is phorphorylated serine) or the HAP binding domain of salivary agglutinin.

Conjugation of sphingosine compounds to proteins and peptides is well-known in the art and is described, e.g., in <CIT>.

One aspect of the disclosure provides the use of a sphingosine compound for coating bone or tooth. The sphingosine compound acts as a protective barrier against, e.g., acid erosion and the formation of salt precipitation leading to tartar.

One aspect of the disclosure provides the use of a sphingosine compound for preventing or reducing tooth demineralisation. Tooth enamel naturally undergoes a process of demineralization, which is increased by the presence of acid, e.g., from food, drinks, gastric acid, or produced by bacteria. Tooth demineralisation is the underlying process involved in the development of dental caries, dental erosion and dentine hypersensitivity (herein referred to as tooth demineralisation disorders). Preferably, said tooth demineralization disorder is due to the presence of acid which has not been produced by bacteria, e.g., the disorder is from acidic food, acidic beverages, or gastric acid. Preferably, said sphingosine compounds are used in methods for the preventing or treating tooth demineralisation disorders resulting from the consumption of acidic food or beverage or from gastric acid.

Hydroxyapatite becomes soluble when exposed to acidic environments. Salts, such as calcium-phosphate salts, may also precipitate on hydroxyapatite.

In preferred embodiments of the methods and products described herein, a sphingosine compound is used together with hydroxyapatite (nano) particles. Hydroxyapatite adheres to the surfaces of teeth and promotes their recalcification and strengthening. It has been successfully used in a dental fine filling method for protecting and restoring pits, fissures and lesions in enamel. Hydroxyapatite has been used in toothpastes in Japan since <NUM> and is commercially available under such names as, e.g., Apagard® nHAP toothpaste, Sangi Co. , Japan, which provides <NUM>% and <NUM>% hydroxyapatite nanoparticle containing toothpaste.

The average particle diameter of the hydroxyapatite particles is preferably in the range from <NUM> to <NUM>, more preferably from <NUM>-<NUM>. The basic building blocks of enamel are usually <NUM>-<NUM> of HAP. Preferably, the hydroxyapatite particles used herein have an average particle diameter of between <NUM>-<NUM>. Most preferred are those have an average particle diameter of around <NUM> as described in <NPL>).

The hydroxyapatite nanoparticles may be provided coated with one or more sphingosine compound as described herein. Such pre-coated particles may be used in oral care compositions or as coating for prostheses, e.g., prosthetic tooth.

In dental caries, acidic end products of anaerobic bacterial metabolism in the dental plaque cause local dissolution of dental enamel, typically at sites which are difficultly accessible for dental hygiene. Dental caries include arrested dental caries, incipient dental caries, pit and fissure cavity, primary dental caries, secondary dental caries, smooth surface cavity. In some embodiments, the tooth demineralization disorder is not dental caries. Dental erosion is the chemical dissolution of dental surfaces by acids from dietary or gastric origin. Dentine hypersensitivity is the pain or discomfort arising from exposed dentine. In preferred embodiments, the compounds described herein are useful for preventing, treating or reducing acid erosion of the teeth, i.e., dental erosion. See <CIT> for a discussion of the development of dental caries and erosion following demineralisation.

Tooth demineralization and tartar formation can be reduced or prevented by the application of a sphingosine compound as described herein.

Remineralization is a natural process resulting in the return of minerals to the tooth surface. The reduction or prevention of tooth demineralization, as used herein, refers to the slowing of the demineralization process such that the net effect of demineralization/mineralization process is such that demineralization is reduced or prevented. Use of a sphingosine compound or composition comprising said sphingosine compound as described herein reduces the net effect of demineralization by at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM>% in comparison to controls. Use of a sphingosine compound or composition comprising said sphingosine compound as described herein also reduces the formation of tartar by least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM>% in comparison to controls.

The sphingosine compounds are also useful in the treatment or prevention of xerostomia. Xerostomia, also known as dry mouth, refers to the lack of saliva and can be caused by insufficient production of saliva. It may be a symptom of an underlying disease such as diabetes and auto-immune disorders or a side-effect of medication. It is also associated with old age, bodily dehydration, and anxiety. Although the sphingosine compounds may be provided in any composition disclosed herein, they are preferably provided in a chewing gum or artificial saliva product for this indication.

The sphingosine compound may be used in a subject suffering from or at risk of suffering from, dental erosion, dental caries, tartar formation, dentine hypersensitivity, and xerostomia. The compound may reduce or prevent said disorders or alleviate a symptom thereof. Alternatively, the compound may be used prophylactically to prevent the risk of developing such a condition or to strengthen the teeth by reducing demineralization.

The sphingosine compounds may be provided in any number of suitable compositions including a pharmaceutical composition, an oral care composition, and food compositions. Preferably, said composition comprises, in particular when phytosphingosine-phosphate is used, a non-ionic, neutral detergent, such as Tween <NUM>, Triton X-<NUM>, Triton X114, Brij <NUM>, Brij <NUM>, Nonidet P40, octylglycoside and ethoxylated stearyl alcohol.

X114, Brij <NUM>, Brij <NUM>, Nonidet P40, octylglycoside and ethoxylated stearyl alcohol, and any non-ionic detergent as listed in <FIG> are compatible.

Preferably, the sphingosine compound is provided in the form of an oral care composition as described herein. The dental enamel of a tooth surface is contacted with said compound or composition by, for example, brushing the teeth with a dentifrice (such as toothpaste or tooth powder), rinsing with a dentifrice slurry or mouthrinse, or chewing a gum product. Other methods include contacting the topical oral gel, mouthspray, or other form such as strips or films with the subject's teeth and oral mucosa. The compound or composition may be applied directly to the teeth, gums, or other oral surface with a brush, a pen applicator, or with the fingers. Sphingosine compounds may also be provided in a food product. Food products include products for human and/or animal consumption and include both solid and liquid (beverage) products. Preferably, the sphingosine compound is selected from one or more of the following: PHS, PHS phosphate, stearoyl PHS, sphinganine, and sphingosine. Preferable the composition includes PHS and at least one additional sphingosine compound, preferably selected from PHS phosphate, stearoyl PHS, sphinganine, and sphingosine. Preferably the composition comprises PHS and sphinganine.

The sphingosine compounds and compositions comprising said compounds are also useful in cosmetic applications.

The sphingosine compounds and compositions comprising said compounds are useful in both therapeutic applications (e.g., prevention and reduction of tooth caries) and non-therapeutic applications (e.g., cosmetic treatments which reduce tooth discoloration).

As used herein, "tooth" refers to a natural tooth as well as hydroxyapatite containing prosthetic teeth, including an inlay, a crown, dentures, and tooth implants.

The sphingosine compounds may be used in any animal in need thereof, including livestock, household pets or other domestic animals, or animals kept in captivity. Pet care products such as chews and toys may be formulated to contain the present oral compositions. Preferably, the compounds are used in humans.

The sphingosine compound is preferably provided to the teeth at least once per day, more preferably twice per day, e.g., once in the morning and once in the evening. Preferably, the sphingosine compound is provided to the teeth regularly, or rather on a daily (or twice daily) basis, over the course of several days, weeks, or months.

Preferably, the sphingosine compound is provided in an oral care composition. Such compositions must therefore be suitable for use in humans and animals. As used herein, oral care compositions are retained in the oral cavity for a time sufficient to contact the teeth and are not intentionally swallowed for purposes of systemic administration. Preferred oral care compositions include toothpaste, dentifrice, tooth powder, tooth gel, subgingival gel, mouthrinse, artificial saliva, denture product, mouthspray, lozenge, oral tablet, or chewing gum. Sphingosine compounds may also be incorporated onto strips or films for direct application or attachment to oral surfaces. Some sphingosine compounds are more effective when provided in a buffer not containing, or containing a minimal amount of, phosphate (e.g., sphinganine in <FIG>). The efficacy of these compounds may be improved by providing them in oral care compositions lacking phosphate (or comprising only minimal amounts). Alternatively, or in addition to, such compounds may be provided in a product that does not rely on saliva. For example, a denture product which treats dentures outside the mouth (such as when placed in a cup) would have no or minimal contact with saliva.

Preferably, the sphingosine compound is present in the oral care composition at a concentration of more than <NUM> ug/ml, preferably at least <NUM> ug/ml, more preferably at least <NUM> ug/ml, more preferably at least <NUM> ug/ml and most preferred at least <NUM> ug/ml. Preferably, PHS is present at a concentration of at least <NUM> ug/ml. In the case of a solid oral care composition the sphingosine compound is present at a concentration of more than <NUM>µg/gram.

Preferably the oral care composition further comprises hydroxyapatite particles as described herein. Preferably, the composition comprises between <NUM>-<NUM> wt. %, more preferably between <NUM>-<NUM> wt. % of said particles. Hydroxyapatite nanoparticles are commercially available from, e.g., nanoXIM• CarePaste (Fluidinova, SA). The hydroxyapatite particles may be precoated with sphingosine compound. Such precoating may reduce the amounts of sphingosine compound needed to have an effect.

The composition and means for preparing suitable oral care compositions are well-known in the art. In some embodiments, the products are in the form of dentifrices, such as toothpastes, tooth gels and tooth powders. A skilled person can select the appropriate components of the oral care composition based on the particular sphingosine compound used.

Components of such toothpaste and tooth gels generally include one or more of a dental abrasive (from <NUM>% to <NUM>%), a surfactant (from <NUM>% to <NUM>%), a thickening agent (from <NUM>% to <NUM>%), a humectant (from <NUM>% to <NUM>%), a flavoring agent (from <NUM>% to <NUM>%), a sweetening agent (from <NUM>% to <NUM>%), a coloring agent (from <NUM>% to <NUM>%) and water (from <NUM>% to <NUM>%) as well as an anticaries agent (from <NUM>% to <NUM>% as fluoride ion) and preservatives. Tooth powders, of course, contain substantially all non-liquid components.

Suitable components of the toothpaste disclosed herein include Carbomer <NUM>, apolymer used for thickening and as an emulsion stabilizer; Carrageenan,.

In a preferred embodiment, the disclosure provides a toothpaste comprising a dental abrasive, a surfactant, a thickening agent, a humectant, and a sphingosine compound as described herein.

Suitable dental abrasives include, for example, silicas including gels and precipitates, insoluble sodium polymetaphosphate, hydrated alumina, calcium carbonate, dicalcium orthophosphate dihydrate, calcium pyrophosphate, tricalcium phosphate, calcium polymetaphosphate, and resinous abrasive materials such as particulate condensation products of urea and formaldehyde.

The most common surfactant currently used in toothpastes is sodium dodecyl sulfate (SDS). We have found that SDS greatly reduces the protective effects of the sphingosine compounds. Therefore, SDS should preferably not be used in the oral care compositions, in particular when phytosphingosine is the sphingosine compound. SDS-free toothpastes are commercially available, in which, e.g., glycyrrhizin (Tom's of Maine Clean & Gentle Care Toothpaste™) or Sodium Lauroyl Sarcosinate (Dr. Katz PerioTherapy Treatment Gel™) is substituted for SDS. More preferably, the oral care composition is free of SDS, SLS, and glycyrrhizin. Preferably, the surfactant is a non-ionic detergent such as Tween <NUM> (polyoxyethylene sorbitan monolaurate), Triton X-<NUM>, Tween <NUM>, and other Tween detergents. Additional non-ionic detergents are listed in <NUM>. Preferably, the oral composition does not contain an ionic detergent.

Suitable thickening agents include carboxyvinyl polymers, carrageenan, hydroxyethyl cellulose, laponite and water soluble salts of cellulose ethers such as sodium carboxymethylcellulose and sodium carboxymethyl hydroxyethyl cellulose, polyethyleneoxide, vora hyaluronic acid glucan, gum karaya, xanthan gum, and gum Arabic.

Suitable humectants include edible polyhydric alcohols such as glycerin, sorbitol, xylitol, butylene glycol, polyethylene glycol, and propylene glycol, especially sorbitol and glycerine.

Suitable flavoring agents include oil of wintergreen, oil of peppermint, oil of spearmint, clove bud oil, menthol, anethole, methyl salicylate, eucalyptol, cassia, <NUM>-menthyl acetate, sage, and eugenol.

An exemplary mouthwash composition includes e.g., ethanol (about <NUM>% and about <NUM>% by weight), propylene glycol (about <NUM>% and about <NUM>% by weight), glycerol (about <NUM>% and about <NUM>% by weight) and in lessor amounts, flavouring and coloring agents. The active ingredients of mouthwash compositions are usually alcohol, chlorhexidine gluconate, cetylpyridinium chloride, hexetidine, benzoic acid (acts as a buffer), methyl salicylate, benzalkonium chloride, methylparaben, hydrogen peroxide, domiphen bromide and sometimes fluoride, enzymes, and calcium. They can also include essential oils that have some antibacterial properties, like phenol, thymol, eugenol, eucalyptol] or menthol. Ingredients also include water, sweeteners such as sorbitol, sucralose, sodium saccharin, and xylitol (which doubles as a bacterial inhibitor). As described above, it is preferred that a non-ionic surfactant is included in the mouthwash.

Chewing gum compositions typically include one or more of a gum base (from <NUM>% to <NUM>%), a flavoring agent (from <NUM>% to <NUM>%) and a sweetening agent (from <NUM>% to <NUM>%).

Artificial saliva, also known as a saliva substitute such as Oralube™, is a solution which simulates saliva. Artificial saliva normally contains water and electrolytes (e.g., potassium, sodium, calcium, chloride, phosphate) and may also contain enzymes, cellulose derivatives, and flavouring agents. Suitable formulations are known in the art and are described, e.g., in <CIT> and <CIT>.

Preferably, the sphingosine compound is provided in a food product supplemented with said compound. Suitable food products include dairy products, processed food products, oils, food and/or vitamin supplements, snack products, and beverage products (such as, sport drinks).

Preferably, the sphingosine compound is provided in beverages supplemented with the said compound. Suitable beverages include water, alcoholic drinks (such as beer, wine), soft drinks (such as cola, iced tea, lemonade, fruit punch, sparkling water), fruit juices (such as orange juice, tangerine juice, grapefruit juice, pineapple juice, applejuice, grapejuice, lime, and lemon juice), vegetable juice ( such as carrot drink and tomato drink), hot drinks (such as coffee based beverages, tea, hot chocolate, gluhwein,). Preferably, the sphingosine compound is provided in an acidic food composition, preferably said food composition has a pH of less than <NUM>, less than <NUM>, or preferably less than <NUM>. The pH of soda pop is around <NUM>.

Food products also include animal chow (e.g., dog foods, cat foods) and supplements (e.g., biscuits, chews). Such products are well-known in the art and are described, e.g., in <CIT>; <CIT> and <CIT> and <CIT>.

Preferably, the sphingosine compound is present in the food product at a concentration of more than <NUM> ug/ml, preferably at least <NUM> ug/ml, more preferably at least <NUM> ug/ml, more preferably at least <NUM> ug/ml and most preferred at least <NUM> ug/ml. In the case of a solid food product the sphingosine compound is present at a concentration of at least 1µg/gram, at least <NUM>µg/gram, at least 5µg/gram, or at least 100µg/gram.

As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of" meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.

The word "approximately" or "about" when used in association with a numerical value (approximately <NUM>, about <NUM>) preferably means that the value may be the given value of <NUM> more or less <NUM>% of the value.

The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

Dental enamel consists largely of hydroxyapatite crystals (Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM>. Sintered at high temperatures, hydroxyapatite discs can be produced with physical characteristics (e.g. hardness and density) resembling those of enamel (Anderson et al, <NUM>), which can be used to determine erosive properties of solutions (Jensdottir et al, <NUM>) by measuring weight loss from hydroxyapatite after immersion in an erosive (acidic) solution.

Hydroxyapatite discs, sintered at <NUM> and with a relative density of <NUM>% were obtained from Swerea, Stockholm, Sweden. The lateral and bottom surfaces of the discs were covered with nail polish so that one side remained uncoated. Next, discs were cleaned by sanding with sand paper (3M734 P600) rinsed with demineralised water, dried at <NUM> overnight and weighed to determine the initial mass. Hydroxyapatite discs were placed in the wells of <NUM> well cell culture plates (Greiner bio-one, Frickenhausen, Germany), to which was added <NUM> of phytosphingosine (PHS in various concentrations dissolved in saliva buffer (<NUM> potassium phosphate, <NUM> KCl, <NUM> CaCl<NUM>, <NUM> MgCl<NUM>, pH <NUM>) under gently shaking for <NUM> hours at <NUM>. After three times rinsing with <NUM> saliva buffer to remove unbound PHS, discs were placed in new wells with <NUM> of <NUM> citric acid (pH = <NUM>). After <NUM> minutes citric acid was pipetted off and discs were rinsed <NUM> times with <NUM> demineralized water, dried overnight at <NUM> and weighted. The difference in weight before and after the erosive treatment was taken as a measure for erosion. The experiments were conducted in triplicate and were repeated at least two times. Formation of saliva pellicle on HAP discs was achieved as follows: saliva was collected without conscious stimulation, as described previously (Navazesh, <NUM>). Saliva was cleared from cellular debris by centrifugation at <NUM>,<NUM> for <NUM> minutes. The supernatant (HWS) was collected and used for coating HAP disks with a salivary pellicle. For this purpose, HAP disks were incubated with <NUM> HWS at <NUM>. After <NUM> hrs, disks were rinsed <NUM> times with distilled water to remove unbound protein. Subsequently, the protective effect of PHS on saliva-coated HAP was tested as described above for bare HAP.

We predicted that PHS adsorption onto HAP may give rise to the formation of a protective film that impedes diffusion of polar compounds and modulates microbial adherence. We first verified if PHS indeed binds to HAP. For this, sintered HAP disks, as a model for dental enamel, were treated with PHS at concentrations between <NUM> and <NUM>µg/ml. Since PHS is moderately soluble in water, we conducted the binding experiments in Tris-Tween. PHS is completely soluble in Tris-Tween as was verified by determination of the PHS concentration before and after a <NUM> minutes centrifugation step at <NUM>,<NUM>. Maximal adsorption occurred at concentrations of <NUM>µg/ml PHS and higher. Under these conditions approximately <NUM>µg PHS was adsorbed onto the surface of the disk. After overnight incubation of HAP disks with saliva, followed by incubation with PHS, even higher amounts of PHS adsorbed compared to the control (HAP disks incubated with saliva buffer) (<FIG>).

<FIG> shows the adsorption over time at a fixed PHS concentration. Within one minute already a substantial amount of PHS was adsorbed. Overall, binding followed a biphasic time course, with an initial fast phase reaching equilibrium within <NUM> hr, followed by a gradual increase over the next <NUM> hrs.

HAP discs, after treatment with a variety of agents (see <FIG>) were exposed to an erosive challenge of <NUM> citric acid (pH = <NUM>) for <NUM> minutes. Pretreatment of discs with either bovine serum albumin or with saliva did not result in significant protection. Despite the fact that PHS is poorly soluble in saliva buffer (Veerman et al. , <NUM>) incubation with HAP disks still resulted in substantial protection (<NUM> - <NUM>% less weight loss compared to the untreated control disks) against a subsequent erosive challenge. Pretreatment with PHS resulted in more than <NUM>% protection against dissolution by citric acid relative to the control disks, which were pretreated with saliva buffer alone, or saliva buffer containing DMSO, which was used for preparing the PHS solution. On the other hand, pre-treatment of HAP disks with <NUM>/ml BSA or whole saliva gave little if any protection against a <NUM> minute lasting erosive attack. To examine if the protective effects were caused by precipitation of insoluble PHS aggregates onto the HAP disks, we repeated the experiment with PHS dissolved in Tris-Tween. This produced essentially the same protection, corroborating that the observed protection in saliva buffer was not due to precipitation of insoluble PHS aggregates onto the HAP surface. This suggests that PHS formed a protective coating on HAP which protected against acidic attacks by citric acid.

Next, we tested the minimal concentration at which PHS afforded protection (<FIG> and <FIG>). This revealed comparable protection at <NUM> and <NUM>µg/ml. At PHS concentrations > <NUM>µg/ml, virtually maximal protection was achieved. Further lowering the PHS concentration to <NUM> ug/ml, which is approximately the critical micelle concentration of PHS (Veerman et al, <NUM>), resulted in a steep decrease in protection. Next we tested the duration of the effect (<FIG>). This showed that after <NUM> hour exposure to citric acid, PHS still protected. Pretreatment of discs with the anionic detergent SDS (<NUM>%), a compound which is commonly found in toothpastes, did not protect against subsequent exposure to citric acid (not shown), indicating that the protection was caused by the specific molecular properties of PHS.

To examine the effect of the solvent used for preparation of the PHS stock solution, PHS was dissolved in DMSO and ethanol to a concentration of <NUM>/ml. These stock solutions were <NUM>-fold diluted in saliva buffer, with or without sonication. Subsequently HAP disks were incubated for <NUM> hr with the resultant working solutions, rinsed and exposed to citric acid. No difference between the various conditions used for preparation of the stock solutions were found (<FIG>). Since dental enamel in situ is covered with a coating of saliva proteins, we tested to which extent this might influence the protection by PHS. We therefore tested the protective effect of PHS on HAP disks that had been preincubated with human saliva, to produce a film of tightly adhering salivary proteins (the salivary pellicle) on the surface of the disks. This revealed that PHS protected saliva-coated HAP to the same extent as it protected uncoated HAP (<FIG>).

To further explore the structural requirements of the observed effects, protection by two other structurally related sphingosines were tested, sphingosine-phosphate and sphinganine (<FIG>). Treatment of HAP disks with sphingosine-phosphate (<NUM>µg/ml saliva buffer) did not afford protection against a subsequent erosive attack by citric acid. Treatment of HAP with sphinganine, which compared to phytosphingosine lacks one hydroxyl group at C<NUM> (<FIG>), protected against citric acid when used in a Tris buffer (<FIG>), but not when used in a buffer containing phosphate ions. Treatment with sphingosine, sphinganine and phytosphingosine-phosphate at <NUM>µg/ml in Tris-Tween produced a protection that was comparable to that by PHS. The other lipids tested, including sphingomyelin, phosphatidylcholine and various N-alkyl sphingosines, did not protect HAP.

We further tested the effect of PHS on the initial bacterial adherence to HAP disks in vitro with S. mutans as a model organism. PHS-coated disks and control disks were submerged in a suspension of S. mutans and after <NUM> hrs the number of adhered bacteria was determined by plating. This revealed a ><NUM>-fold decrease in number of adhered bacteria to PHS-coated HAP, compared to base HAP (<FIG>)).

HA disks were pretreated with buffer or <NUM> ug/ml PHS for <NUM> hours (<FIG>, left two bars) followed with a treatment with <NUM> citric acid (pH=<NUM>) for <NUM> minutes. PHS pre-treatment protects against the erosive effects of citric acid. HA disks were pretreated with either buffer or <NUM> ug/ml PHS and then exposed to <NUM> citric acid in the presence of <NUM> ug/ml PHS for <NUM> minutes (<FIG>, right two bars). PHS present in an erosive fluid protects against erosion even with untreated HAP disks. (PHS has no effect on the pH of citric acid.

Six different sphingolipids including PHS, PHS phosphate, Sphingosine, Sphinganine, Stearoyl PHS and Sphingomyelin were tested for their antifouling properties against two primary colonizers i.e.- Streptococcus sanguinis and Streptococcus gordonii and a late colonizer, Streptococcus mutans.

HA discs were incubated O/N with <NUM>µg/ml lipid at <NUM>. Subsequently, the discs were washed to remove unbound lipid. Then the lipid-coated HA discs were incubated for <NUM> hr with <NUM> of ~<NUM> cells/ml. After washing, the adherent bacterial cells were desorbed by sonication and transferred to agar plates. After <NUM> hr CFUs were counted. mutans PHS phosphate and Stearoyl PHS showed clear antifouling activity by almost <NUM> log CFU/ml. Sphinganine showed a <NUM> log reduction (<FIG>).

sanguinis, Sphingomyelin showed reduction in bacterial adherence by <NUM> log value while Sphinganine exhibited reduction by <NUM> log value (<FIG>). In case of S. gordonii, PHS phosphate and Sphinganine showed reduction by <NUM> log values (<FIG>).

Phytosphingosine (from Doosan Corporation, France) was a kind gift of Dr P. Ekhart (Innopact BV) Sphinganine, <NUM>-hydroxysphinganine-<NUM>-phosphate, N-acetoyl <NUM>-hydroxysphinganine , N-octanoyl <NUM>-hydroxysphinganine, N-stearoyl <NUM>-<NUM>-hydroxysphinganine (all from Saccharomyces cerevisiae), sphingomyelin and sphingosine were obtained from Avanti Polar Lipids (Alabaster, Alabama). Di-myroistyl phosphatidylcholine was obtained from Sigma-Aldrich. Hydroxyapatite disks (diameter <NUM>, height <NUM>), sintered at <NUM> and with a relative density of <NUM>% were obtained from Swerea, (Stockholm, Sweden). HAP disks manufactured in this way have physical characteristics (e.g. hardness and density) resembling those of enamel (Anderson et al, <NUM>) and can be used to determine erosive properties of solutions.

Protection of HAP against erosion was determined essentially as described by Jensdottir et al, (<NUM>), by measuring weight loss from HAP disks before and after immersion in an erosive (acidic) solution using a Sartorius GD503 analytical precision scale. For the erosion experiments, the lateral and bottom surfaces of the disks were covered with nail polish leaving the circular upper surface uncoated. Next, disks were cleaned by sanding with fine sand paper (3M734 P600), rinsed with demineralised water, dried at <NUM> overnight and weighed to determine the initial mass. Stock solutions of lipids were prepared in ethanol to a concentration of <NUM>/ml. These stock solutions were diluted further to the desired concentration in the working buffer. Working solutions of PHS were prepared in saliva buffer (<NUM> potassium phosphate, <NUM> KCl, <NUM> CaCl<NUM>, <NUM> MgCl<NUM>, pH <NUM>) or in <NUM> Tris (pH <NUM>) supplemented with <NUM>% Tween <NUM> (Tris-Tween). HAP disks were placed in the wells of <NUM> well cell culture plates (Greiner bio-one, Frickenhausen, Germany), to which <NUM> of the lipid solutions was added and incubated under gentle shaking for <NUM> hours at <NUM>. After three times rinsing with <NUM> of the incubation buffer to remove unbound lipid, disks were incubated with <NUM> of <NUM> citric acid (pH = <NUM>). After <NUM> minutes incubation at <NUM> under gentle shaking, citric acid was pipetted off and disks were rinsed <NUM> times with <NUM> demineralized water, dried overnight at <NUM> and weighed. The difference in weight before and after the erosive treatment was taken as a measure for erosion. All incubations were conducted in triplicate and each experiment was repeated at least two times.

Formation of saliva pellicle on HAP disks was achieved as follows: saliva was collected without conscious stimulation, as described previously (Navazesh et al. HAP disks were incubated with <NUM> human whole saliva (HWS) at <NUM>. After <NUM> hrs, disks were rinsed <NUM> times with distilled water to remove unbound protein. Subsequently, the protective effect of lipids on saliva-coated HAP was tested as described above for uncoated HAP disks. The study was approved by the Institutional Ethical Board of the Academic Hospital Vrije Universiteit at Amsterdam and informed consent was obtained from the donor.

PHS was dissolved in concentrations ranging from <NUM> to <NUM>µg/ml in <NUM> Tris buffer supplemented with <NUM> % Tween <NUM> (pH <NUM>) (Tris-Tween). Tween <NUM> was added to keep PHS in solution. Disks were incubated with the PHS solutions for <NUM>, and then rinsed with demin water (<NUM> times) to remove unbound PHS. HAP bound PHS was extracted by incubation with <NUM> methanol for <NUM> minutes. Since nail polish dissolved in methanol, in the binding experiments HAP disks were used without nail polish. It was verified that no PHS was adsorbed to the plastic, by conducting control incubations without disks. After evaporation, the residue was dissolved in <NUM>µl of methanol. To <NUM>µl of this solution 25µl ortho-phtaldialdehyde reagent (OPA, Sigma-Aldrich) to enable fluorimetric quantification of bound PHS. Fluorescence was measured on a Fluostar microplate reader at excitation/emission wavelengths of <NUM>/ <NUM>. Absolute quantities were determined by reference to a standard curve of PHS. All incubations were conducted in triplicate and the experiment was repeated two times.

Claim 1:
Use of a sphingosine compound having formula I:
<CHM>
wherein X is a alkyl or alkenyl having <NUM>-<NUM> carbons, preferably <NUM> to <NUM> carbons, more preferably a linear alkyl;
R<NUM> is selected from H and C(=O)C<NUM>-<NUM>, preferably R<NUM> is H;
R<NUM> is selected from H and OH, preferably R<NUM> is OH; and
R<NUM> is selected from phosphate, OH or F, preferably OH,
or a conjugate of said compound, for the non-therapeutic cosmetic prevention of discoloration of a tooth.