NZ721687B2

NZ721687B2 - Stabilized stannous compositions

Info

Publication number: NZ721687B2
Application number: NZ721687A
Authority: NZ
Inventors: Eric Charles Reynolds
Original assignee: The University Of Melbourne
Priority date: 2013-12-24
Filing date: 2014-12-24
Publication date: 2021-03-02

Abstract

The present invention relates to improved complexes of amorphous calcium phosphate and/or amorphous calcium fluoride phosphate stabilised by phosphopeptides/phosphoproteins by addition of stannous ions. These complexes have anticariogenic properties useful to protect tooth structures as they remineralize (repair) early stages of dental caries and have other dental/medical applications (including anti-calculus, anti-erosion/corrosion and anti-dentinal hypersensitivity). Methods of making the complexes of the invention and of treatment or prevention of various dental conditions including dental caries, dental calculus, dental erosion/corrosion and dental hypersensitivity are also provided. alize (repair) early stages of dental caries and have other dental/medical applications (including anti-calculus, anti-erosion/corrosion and anti-dentinal hypersensitivity). Methods of making the complexes of the invention and of treatment or prevention of various dental conditions including dental caries, dental calculus, dental erosion/corrosion and dental hypersensitivity are also provided.

Description

Stabilized Stannous Compositions Field of the invention The. present ion s to improved complexes of amorphous calcium phosphate and/or ws calcium fluoride phosphate stabilised by phosphopeptideslphosphoproteins by addition of stannous ions. These complexes have antioariogenic properties useful to protect tooth structures as they ralize (repair) early stages of dental caries and have other dentalimedicai applications (including anti— celculus, anti—erosionicorrosion and anti~dentinal ensitivity). Methods of making the complexes of the invention and of treatment or prevention of various dental 1O conditions including dental caries, dental calculus, dental nfcorrosion and dental hypersensitivity are also provided.

This application claims priority to Australian patent application nos. 2013905081, 2014901202" and 2014903815, these applications are orated by reference in their entirety.

Background of the invention Common causes of hypomineralized s are caries and iluorosis.

Dental caries is initiated by the deminerelizetinn of hard tissue of the teeth usually by organic acids produced from fermentation of dietary sugar by dental plaque odontopathogenio bacteria. Dental caries is still a major public health problem.

Further, restored tooth surfaces can be susceptible to further dental caries around the margins of the restoration. Even though the prevalence of dental caries has decreased through the use of fluoride in most developed, countries, the disease remains a major public health problem. Dental erosion or corrosion is the. loss of tooth mineral by dietary or regurgitated acids, Dental hypersensitivity is due to exposed dentinal s through loss of the protective mineralized layer, cementum. Dental us is the unwanted accretion of calcium phosphate ls on the tooth surface. All these conditions, dental caries, dental erosion, dental hypersensitivity and dental calculus are ore imbalances in the level of calcium ates.

Enamel fluorosis (mottling) has been recognized for nearly a century; however, the 3O aetiological role of fluoride was not identified until 1942 (Black and McKay 1916). The characteristic appearance of fluorosie may be differentiated from other enamel [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r disturbances (Fejerekov etat, 1991), The clinical es of fiuorotic lesions of enamels (FLE) represent a continuum ranging from fine opaque lines following the perikymate, to chalky, white enamel, (Fejerskov et at, 1990; Giembro et all” 1995). The ce of a comparatively highly- mineralized enamel outer surface and a hypominerelized subsurface in the fluorotic lesion simulates the incipient enamel “white spot" carious lesion (Fejerskov et 81., 1990). With sing ty, both the depth of enamel invoived in the lesion and the. degree of hypomineralization increases (Fejerskov et at, 1990, Giambro et at, 1.995). The development of tiuorosis is highly dependent on the dose, duration and timing of fluoride re (Fejersk'ov at at, 1990, Fejerskov et at, 1998; Aoba and Fejerskov, 2002) and is believed to be related to elevated serum fluoride concentrations. Chalk-y “white spot” s may aiso form on developing teeth in en such as after treatment with antibiotics or fever. Such lesions indicate areas of hypomineralization of the tooth .

Depending on lesion severity, fluoroeis has been managed clinically by restorative ement or micro~abrasion of the outer enamei {Den Besten and Thariani, 1992; Fejersltov et at, 1996). These treatments are unsatisfactory because they invoive ations or removal of tooth tissue What is desired is a, treatment that will mineralize the hypomineralized enamel to produce a natural appearance and structure.

Specific complexes of casein phosphopeptides and amorphous calcium phosphate 2E] (“OPP-ASP", ble commercially as Rec-aldentm) have been shown to remineralize enamel subsurface s in vitro and in situ (Reynolds, 1998; Shen et all! 2001; Reynolds etai. 2003).

WC) Q8t40406 in the name of The University of Melbourne (the contents of which are herein incorporated fully by reference) describes casein phosphopeptideuemorphous calcium phosphate complexes (CPP—ACP) and OPP—stabilised amorphous calcium fluoride phosphate complexes (CPP-ACFP) which have been produced at alkaline pH.

Such compiexes have been shown to prevent enamel deminereliz-ation and promote reminerelization of enamel subsurface s in animal and human in situ caries models (Reynolds, 1998); The CPP which are active in forming the complexes» do so whether or not they are part of a full—length casein protein. Examples of active (CPP) that can be isoieted after tryptic digestion of full length casein have been specified in US Patent No. 5,015,628 and include es Bos oer—casein X»5P (ft-3979) {1], Bee Ei~ casein-4P (f1 ~25). [2], Bee egg-casein X-4P (ME-70) [33 and 80’s egg-casein X—4P (f‘l- [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar 21) [4]. Moreover, improvements on these compositions are disclosed in WC) 20061056013 and W0 2007(090242 and specific uses (the contents of which are herein incorporated fully by nce) us fluoride (SnFQ) is one of a number of metal fluoride salts that has been proposed as an antioaries and antiplaque agent r there are difficulties in delivering this agent in toothpaste, mouthwash and other oral care products due to the» instability of the stannoue ions. in the presence of hydroxide ions and phosphate ions etannous can precipitate as stannou's hydroxide and stannous phosphate complexes.

This precipitate is. significantly less biologically available and can be more easily ' removed from the oral cavity by swallowing. Furthermore the stannous ions can be oxidized to ‘Stannio ions (SnIV) which are. even more reactive to produce poorly soluble forms with very low activity. New toothpaste formulations with 5an rely on being non— aqueous or use polyphosphate to ize the stannous ions. However, these formulations have tow acceptance particularly by people with dry mouth. There is a need to provide improved or alternative treatments for hypomineralized lesions.

Reference to any prior art in the ication is not, and should not be taken as, an ledgment or any form of tion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a 2t] person d in the art.

Summary of the invention in one , the invention provides a stannous—assooiated stabilized amorphous calcium phosphate (ACP) and/or ous calcium fluoride phosphate (ACFP) The astannous’ may be bound to the stabilized amorphous calcium ate (ACP) and/or amorphous calcium fluoride phosphate (ACFP‘) as determined using the experimental protocol in Example 2. In one em bodiment, stannous—assooiated stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) complex are produced by the method as described herein, including but not d to the method described in Example 1.

Without being bound by any theory or mode of action, it is believed that phosﬂoeptides, such as casein phosphopeptides, can stabilize a stannous nd in an aqueous environment and in the presence of stabilized amorphous calcium [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar phosphate (ACP) and stabilized amorphous calcium fluoride phosphate (ACFP) these xes are superior to other forms of fluoride and ized ACP or ACFP in relizing enamel subsurface. lesions. Mineralization of dental surfaces can be significantly enhanced by providing a stannous compound during the process of mineralization by stabilized ACP andior stabilized ACFP. in particular, it has been found that the mineralization of enamel by stabilized soluble forms of stannousessooiated .ACP compiexes and stannous-associated ACFP com plexes is enhanced ed with stabilized ASP and fluoride without. associated stsnnous. in other words, the stannous ions complex with CPP~ACP and/or FP complexes, and these Sn~associatecl 1t] CPP—AC(F)P complexes then deliver superior properties. Various. compositions incorporating these complexes for administration are useful. Where the fluoride stennous salt is used. additional fluoride ions are available in itions of the stennous—a’ssooieted A'CP/ACFP complexes. Additional fluoride ions may also be provided by inclusion of NeF in the composition.

The invention also provides a stannous-assoolated stabilized ACP or .ACFP having a stennous ion t of at least 1 mole of us per mole of phosphooeptide.

Preferably. the stannous-assooisted stabilized ACP or ACFP has a stennous ion content of at least 2, .3, 4, 5, 8, 7, 8, 9 or 10 moles of stannous per mole of phosphopeptide. Even more preferably, the stanno‘us ion content is in the range of 1 to 2E) 100, 1 to 50, ‘l to 20 or 1 to 10 moles of s-tannous per mole of phosphopeptide. The present invention also provides a ”composition sing, or consisting of, stannous- associate-d stabilized ACP and/or ACFP xes as described . in any embodiment the sta‘nnous ion t above may be the stann‘oue ion content tightiy—oound to the complex (as described herein). In assessing the» stennous ion content. the tightly-bound stannous ion content is measured by the methods described herein; in particular, in Example 6.

The invention also provides a, stannousessociated stabilized ACP andror ACFP complex comprising stennous ions that remain associated with the complex after oentrifugation, in a 1600 lar weight out off filter at about 30009 for 1 hour at room temperature.

The invention also provides a stannous—assooieted stabilized ACF’ or ACFP having at least ﬁes 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r stannous associated. with the complex as tightly bound as determined by the method in Example 6, The ion also provides a stannousassociated stabltized ACP or ACFP having at least 50, 60,. 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the stan‘nous used in ing the complexes orated into the complexes. The complex may» be prepared as outlined in Example t.

In one embodiment the invention provides a stannous fluoride associated stabilized amorphous calcium phosphate (ACP) andior a us fluoride associated amorphous calcium fluoride phosphate (ACFP) comptex‘. In another aspect, invention provides a composition including a stannous compound and stabilized ACP or ACFP. in either embodiment, the stannous compound is preferably stannous fluoride andlor stannous chloride. Optionally, NaF is also included, in one aspect, the present invention provides a method of mineralizing a dental surface or sub-"surface comprising contacting the dental surface or subsurface with (a) a composition including a stannous compound and stabiiized ACP or ACFP, or (b) a stannous-associated stabilized ACP or ACFPi The stannous containing compound can be any soluble stannous containing compound suitable for oral use. Preferably, stannous containing compound is a stannous salt. The stannous salt may contain fluoride. A stannous salt includes, but not limited to, stannous fluoride, stannous- chloride, potassium stannous de, sodium stannous fluoroziroonate, stannous chloride fluoride, stannous acetate, sodium stannous fluoride, stannous uorozlrconate, stannous sulfate, stannous te, stannous ate, um monostannous citrate. Preferred us salts include us fluoride and stannous chloride The dental surface is aloiy dental enamel or dentine. in one embodiment the dental surface is a lesion in the enamel, such as a lesion caused by , dentai erosion or fiuorosis. in another embodiment the surface is exposed dentine causing dentinal ensitivity.

Preferably, the stabilized amorphous calcium ate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) is phosphopeptide stabitized. Preferably, the phosphopeptide (as defined below) is a casein phosphopeptide, [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r ed set by r [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar in a preferred embodiment, the phosphopeptide stabilised amorphous calcium phosphate (ACP) or amorphous caicium fluoride phosphate (ACFP) complex has tightly bound and loosely bound calcium, wherein the bound calcium in the complex is less than the tightly baund calcium in an ACP or ACFP complex formed at a" pH of 7.0.

Optionally, the ACP or ACFP is predominantly in a basic form, in another aspect the invention also provides a composition ing stannous- associated stabilized ACP or ACFP, further comprising fluoride, wherein fluoride is provided as stannous fluoride and Sodium fluoride. Preferably, the composition es 2% w/v stabilized ACP or ACFP, about itO’Oppm fluoride as stannous de and ' about 350ppm fluoride as sodium fluoride, or the composition includes 0.4% wlv stabilized ACP or ACFP, about 220ppm fluoride as stannous fluoride and about 70ppm fluoride as sodium fluoride. Preferably, the composition is a toothpaste. in a preferred embodiment, the calcium ion content of the stabilised ACP or ACFP complex is in the range of about 30 to 100 moles of calcium per mole of PP. More preferably, the caicium ion content is in the range of about 30 to about 50 moles of calcium per mole of PP. in a preferred ment the ACP andior ACFP is in the form of a casein pheaphopeptide stabilized ACP and/or ACFP complex.

Preferably, the phase of the ACP is primarily (i.e. $096) .a basic phase, wherein the ACP comprises predominantly the species Ca2+, P04} and OH". The basic phase of ACP may have the general formula [CagtP04)g]x[Ca2(POi)(OH)] where x 2 ‘l. Preferably x = 1—5. More preferably, x = i, i.e. the two components of the formula are present in equal proportions. ingly, in one embodiment, the basic phase of ACP has the formula 083(PO4)2082(PO4)(0H).

Preferably, the phase of the ACFP is a primarily {i.e. >50%) basis phase, wherein the ACFP comprises predominantly the s Ca”, PO43‘ and F‘. The basic phase of ACFP may have the general a [C33(PO¢$)2]x[Caz(PO4)F]y where x a 1 when y = 1 or where y 2 1 when x = 1. Preferably, y = 1 and x = 1—3. More preferably, 3/ = i and x = t, i.e. the two components of the formula are present in equal proportions.

Accordingly, in one embodiment, the basic phase of ACFP has the formula Ca3(PO4)2C;a3(P04)F.

[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar lo one embodiment, the ACP complex consists essentially of phosphopeptides, calcium, phosphate and hydroxide ions and water. in one embodiment, the ACFP complex consists essentially of phosphopeptides, calcium, phosiohate, ﬂuoride and hydroxide ions and water, In a further aspect of the present invention there is provided a method of mineralizinsg a dental surface comprising providing (a) a composition including a stahnous compound and stabilized ACP or ACFP, or (b) a stannous-associated stabilized ACP or ACFF’. in a preferred embodiment the dental e is enamel. in a further aspect of the t invention there is provided a method of g a layer on the surface of a tooth comprising providing (a) a composition including a stannous compound and stabilized ACP or ACFP, or (b) a stannous-associ-ated ized ACP or ACFP, Preferably the tooth is in a t identified as being susceptible to, or suffering from, any one or more of dental erosion, demineraiization, caries or dentinal hypersensitivity. Preferably, the stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) is phosphopeptide stabilized.

Preferably, the phosphopept’ide (as defined below) is a casein opeptide.

Such a layer may be characterised has a calcium : phosphate ratio lent to normal apatite, preferably where the ratio is about 2:1. The layer ideally contains an amount of calcium that is about 20 wt%. 2O Preferably, the layer has about 3 to 12 told more stannous ions that sound enamel.

Preferably, the layer contains carbon, oxygen, fluoride, phosphate, calcium and stannous, The layer may t about an eiementai analysis of 20-30wt% carbon, 35— eﬁwtﬂxii oxygen, O.‘l-1‘wt% fluoride, 8 to 12wt% phosphate, 16 to .24wt% calcium andior t% stannous. Alternatively, the layer may t an elemental analysis of any one of the elements, such as calcium, ate, de, carbon andfor us, as shown in any one of Table 3 or 4. ln a further aspect of the present ion there is provided a process of forming a layer having a m : phosphate ratio at, or near, that of normal apatite on a dental surface, the process sing contacting the dental surface with (a) a composition inciuding a stanno‘us compound and stabilized ACP or ACFP, or (b) a stennous— assoc‘ d stabilized ACP or ACFP.

[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by r A method of protecting a tooth surface comprising providing (a) a composition inciudin-g a stannous compound and stabilized ACP or ACFP, or (lo) a stamens—associated stabilized ACP or ACFP. Typically the tooth surface may be one that has been fied as benefiting from a surface iayeri for e, due to an increased iikelihood of demineralization. The tooth surface may be an enamel surface or dentin‘e surface.

In a further aspect of the present invention there is provided a method for treating; fluorosis sing contacting a fiuorotic iesi‘on in tooth enamel with (a) a composition ding a stannous compound and stabilized ACP or ACFPV or (be) a stannous— associated stabilized ACP or ACFP. in a further aspect of the present invention there is provided a method for ng dental caries comprising contacting a caries tesion in tooth enamel with (a) a composition inciuding a stannous compound and stabiiized ACP or AGFP, or (b) a. stannoue— associated stabilized ACP or ACFP‘.

In a r aspect of the: t invention there is provided a method for treating dentai n comprising contacting a iesion in tooth enamel caused by erosion with (a) a, composition including a stannous compound and stabilized ACP or ACFP, or (b) a stannous—associated stabilized ACP or ACFP. in a further aspect of the present invention there is ed a method for reducing dentinai hypersensitivity comprising contacting exposed dentine with (a) a composition including a stannous compound and stabiiized ACP or ACFP, or (b) a stannous— associate-d stabilized ACP’ or ACFP. lo a further aspect of the present invention there is provided a method for remineralizing a lesion in tooth enamel or dentine comprising ting the lesion with (a) a. composition ing a stannous compound and stabilized ACP or ACFP, or (b) a stan‘nous-associated stabilized ACP or ACFP.

Preferably the s-associated stabilized ACP or ACFP are stabilized by a phosphopeptide (PP). In a red embodiment the phosphopeptide is a casein phosphOpeptides. Preferably, the ACP or ACFF’ is in the form of a casein phosphopeptide stabiiized ACP or ACFP complex. ln any aspect or embodiments as described herein: the stabilized ACP andfor ACFP or stannﬁ—associated stabilized ACP or ACFP may be in a formulation with additionai [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar ed set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar calcium phosphate. Typically, the ation es a PP stabilized ACP andfor ACFP compiex together with at least an equal amount by weight of calcium phosphate. in a further aspect of the invention there is provided a method for remineraiizing a lesion in tooth enemei comprising contacting the lesion with (a) a composition including a stan‘nous compound and stabilized ACP or ACFP, or (b) a stannous-associated stabilized ACP or ACFP followed by administering a composition containing sodium bicarbonate or urea. ably, the composition is a mouthrinse or mouthwash containing sodium bicarbonate or urea, in any aspect or embodiment of the ion bed herein, a compound which is capabie of increasing or maintaining the pH of a solution may be: administered concurrently with, as a atment to, or as a poet—treatment to (a) a composition including a stannous compound and stabiiized ACP or AGFP, or (b) a. stannous— associated stabilized ACP or ACFP‘. in any aspect or embodiment of the invention described herein, (a), a composition inciuding a stannous compound and ized ACP or ACFP, or (b) a stannous— associated stabilized ACP or ACFP may be applied to the mouth, tooth or lesion by the subject in need of treatment or by a dentai health care professional.

A composition including a us compound and stabilized ACP or ACFP‘ may be ted with the dental surface for a period of about 1 to 60 minutes, or for about 1 to minutes. in one embodiment, a composition inciuding a stannous nd and stabilized ACP f ACFP is contacted with, the dental surface for about 20 minutes.

Preferably a composition including a stannous compound and ized ACP or ACFP is contacted with the dental surface for a period of about 1 minute to 2 hours, or 5 minutes to 60 minutes or about 10 minutes. The composition including a st‘annous compound and stabilized ACP ! ACFP may be repeatediy applied to the dentai surface over a period of '1 day to several months.

A stannous—associated ized ACP or ACFP may be contacted with the dental surface for a period of about 1 to 60 minutes, or for about 1: to 30 minutes. In one embodiment, a stannous—associated stabilized AC? 1 AGFP is contacted with the dental surface for about 2.0 minutes. surfa Prefeﬁ: a stannous-associated stabilized ACP or AC-FP is contacted with the dentai r a period of about 1 minute to 2 hours, or 5 s to 80 minutes or about [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by r [Annotation] r MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar minutes. The stennous-associeted stabilized ACP or ACFF’ may be repeatedly applied to the dental surface over a period of 1 day to several months. in one embodiment, (a) a Composition including a stannous compound and stabilized ACP or ACFP, or (b) a usassociated stabilized ACP or ACFP, is contacted with; the dental surface 1 to 60 minutes, or 1 to 30 minutes, OH to 5 minutes. in one embodiment, the dental surface is in need of such treatment. Therefore the invention es in addition to the steps of any method described herein a step of identifying a subject ing fiuorosis, dentai caries, dentinel hypersensitivity or dental calculus, a white spot lesion; 3 tic lesion; a caries lesion; or a lesion caused by tooth erosion, or dental plaque or gingivitis or periodontitis.

The present invention provides a composition for iizing a dental surface or sub- surface that is also capable of reducing plaque, gingivitis and periodontitis sing (a) a composition including a stannous nd and stabilized ACP or ACFP, or (b) a stamens—associated stabilized ACP or ACFP. in a r aspect, there is provided a method of treating or preventing one or more of each of dental caries, tooth decay, dental erosion and fluorosis, comprising the steps of administering (a) a composition including a stennous compound and stabilized ACP or ACFP, or (b) a stannous‘associateci stabilized ACP or ACFP, thereby treating or preventing one or more of each of dental caries, tooth decay, dental erosion and fluorosis. Topical administration of the complex is preferred. The method preferably includes the administration of the complex in a formulation as described above.

The present invention also provides a composition containing a stannouseseocieted stabilized ACP or ACFP. Preferably, the composition further includes a pharmaceutically acceptable carrier, diluent or exci'pient. in a preferred embodiment, the phosphopeptide stabiiised ous calcium phosphate (ACP) or amorphous calcium de phosphate (ACFP) complex in the composition has tightly bound and loosely bound calcium, wherein the bound calcium in the compiex is less than the tightly bound calcium in an ASP or ACFP complex formed at a pH of 7.0. Optionally, the ACP or ACFP is inantly in a basic form. in another preferred embodiment, the calcium ion content of the ised ACP or ACFP . mplex in the composition is in the range of about 30 to too moles of calcium [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar per mole of PP. More preferably: the oaloium ion content is in the range of about 30 to about 50 motes of calcium per mole of PP. in any ment as described , the ACP andi'or ACFP in the composition can be in the form of a casein phosphopeptide stabilized ACP andiorACFP complex.

The invention also relates to a kit for the treatment or prevention of one or more of dental caries, fluorosis, dental hypersensitivity and dentai n ing: (a) a nd capable of increasing or maintaining the pH of a solution! and (b) a composition including a stannous compound and stabilized ACP or ACFP, or (o) a stannous—assooiated stabilized ACP or ACFP.

Preferably, the stennous—assooiated stabilized ASP" or ACFP is in a pharmaceuticaliy acceptable carrier. Desirably, the kit further includes instructions for their use for the mineralization of a dental surface in a patent in need of such treatment. The instructions may describe the use of the kit to treat or prevent one or more of each of dental caries, tooth decay, dental erosion, dental hypersensitivity and fluorosis. in one embodiment, the agent and the complex are present in suitable amounts for treatment of a patient.

The. ition or hit of the invention may further include a source of fluoride ions. The fluoride ions may be from any suitable source. A source of fluoride ions may include free ﬂuoride ions or fluoride salts. Exampie's of s of fluoride ions include: but are not limited to the following: sodium ﬂuoride, sodium monofluorophosphate, us fluoride, sodium silicofluoride and amine ﬂuoride. These may be provided in solution (typically an aqueous on), or a suspension.

Brief description of the drawings Figure 1: entative SEM images of enamel subsurface iesions before (A) and after (8) treatment with stabilized Sani’ACP also demonstrating the surface layer.

Figure 2: entative SEM images of enamel subsurface tesions before (A) and after (8) treatment with stabilized N-aF/ACP- [Note no ion ot'a novel surface layer].

Figure 3: Scanning Electron Microscopy—Energy Dispersive Spectroscopy (SEM-EDS‘) of the mineraiised surface layer resulting from treatment with stabilized Sani’ACP.

Figure 4: Representative SEM image of eiementai is by SEMI-EDS of the mineined surface layer resulting from treatment with stabilized SanfACP. ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar Figure 5: Representative SEM image of elemental analysis by SEM-EDS of the mineralised surface layer resulting from treatment with stabilized Sani’ACP.

Figure 6: Remineratization of enamel face lesions by stabilized SaniACP versus stabilized NaFIACP. The numerical values of this Figure are shown in Table 2.

Figure 7: In situ remineralization of enamel subsurface s by CPPACP SanlNaF ed with CPP-ACP, San and NaF, NaF with or without P.

Detailed ption of the. embodiments it will be tood that the invention disclosed and defined in this specification extends to alt alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative s of the invention.

Further s of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the acoom g drawings.

Reference will now be made. in detail to certain embodiments of” the ion. While the invention will be described in conjunction with the embodiments, it wili be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and lents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present ion.

The present invention is in no way limited to the methods and materials described.

All of the patents and publications referred to herein are incorporated by reference in their entirety.

For purposes of interpreting this specification, terms used in the singular will also include. the piurat and vice versa, As used herein, except where the t requires vise, the term ”comprise" and variations of the term, such as "comprising“, “comprises" and “comprised", are not intended to exclude further additives, components, integers or steps. As used herein, except where the context requires otherwise, ”comprise” and “include” can be used interongeably.

[Annotation] r None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar The present invention is based on the finding that casein phosphopeptides can ize a stannous compound, such as San‘, in an aqueous environment and in the presence of stabilized amorphous calcium phosphate (ACP) and stabilized amorphous calcium fluoride phosphate (ACFP‘) these formulations are Superior to other forms of fluoride and stabilized ACP or ACFP in remineralizing enamel subsurface lesions. Mineralization, of dental surfaces can be significantly enhanced by providing a stannous compound during the process of mineralization by stabilized ADP and/or stabilized ACFP. in particular, it has been found that the lization of enamei by ized soluble forms of stannous‘associated ACP complexes and usassociated ACFP 1t] complexes is enhanced ed with Stabilized ACP and fluoride Without associated stannous, The stannous ion in the presence of stabilized ACP andior stabilized ACFP can produce a protective surface layer on the /dentine to help prevent dental caries, dentinal hypersensitivity, dental plaque and periodontal disease. The layer which. includes stannous ions and stabilized ACP andror stabilized ACFP can provide a reservoir for ca..loium.,. phosphate, and fluoride for mineralization of the tooth surface or face.

These formulations have been designated stannous-assooia’ted stabilized ACP and ous-assooiated stabilized ACFP.

Without being bound by any theory or mode of action it is believed that the stannous 2t] ions are stabilized by the presence of the phosphopeptides, ularly casein phosphopeptides. ore, it is believed. that the phosphopeptides deliver stannous and fluoride ions together with the calcium and phosphate ions to promote remineralization. Reminera‘lization may be a result of formation of calcium and stannous fluorapatite. it is also believed that the stannous ions cross~linit the phosphopeptide stabilized ACP or stabilized ACFP at the tooth surface to form a layer that can protect the tooth surface from demineralization. The presence of the stannous in the surface layer would render it hard and resistant to degradation due to normal wear and tear or other processes such as erosion. A further age of the invention is that the. us ions may assist to kill oral bacteria that produce acid and other metabolic ts which promote demineralization.

Stannous is known to form precipitates with hydroxide ions and phosphate ions thereby reducing the bioavailabiltiy and ty of the stannous ion. The resulting promotion of stabilnl—ACPistabil'izeduACFP driven remineralization by stannous ions is surprising [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar as it would be expected that the hydroxide ions (ed OH“) and phosphate ions (eg P043“): provided by the ized~ACP or stabilized~ACFP would cause precipitation of the stahnous to stannous hydroxide and stahnous phosphate, thereby severely decreasing any activity of the stannous containing nd. Further, it would be ed that formation of a precipitate of stannous ions with hydroxide ions would not onty reduce the bioavailahility of the stannous ions but also remove hydroxide ions from the environment thereby reducing the pH. A reduction in pH would promote dem‘ineralization and hinder mineralization The t invention provides stannous—essocieted stabilized ACP- or ACFP ' complexes and methods of mineralizing a dental surface or sub-surface comprising contacting the dental surface or subsurface with a, stannouseassociated ized ACP or ACFP complexes. A dental subsurface is typically a hypomineralized lesion such that the usassooiated stabilized ACP or ACFP complexes contacted to the dental surface migrate through any surface layer, is. peiiicie andfor plaque, through the porous dental surface to the region requiring mineralization. These stannous~assooiated stabilized ACP or ACFP complexes aiso generate a protective surface layer on the enamelfde’ntine to help prevent dental caries, al hypersensitivity, dental plaque and ontal e The dental surface is preterabty dental enamel or dentine. The dental surface may be a lesion in the enamel, such as a tesion caused by series, dental 2E) erosion or sis. in one aspect the invention provides, a stannous—assooiated stabilized ous calcium phosphate (ACP) or stannoue-assooiated amorphous calcium fluoride phosphate (ACFP) The stannous may be bound to the stabiiized amorphous calcium phosphate {ACP} andlor amorphous calcium fluoride ate {ACFP} as ined using the experimental protocol in Example 2. in one embodiment, the stannous— associated stabilized ACP or stannous—associated stabitized ACFP are produced by a method as described herein, inciuding but not limited to, the method described in Example 1.

The stannous is present in the stannous—associated stabilized amorphous calcium phosphate (ACP) or stannous—assooiated amorphous calcium fluoride phosphate (ACFP) complex by binding to or being incorporated in the complex. This complex associated stannous, wherein the stannous is bound to or incorporated in the complex, can notermine'd using filtration and atomic absorption spectrophotometry. Tightly [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar ed set by wilksar ation] r None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar associated oomptexed stennous is measured as the difference between total stannous less lo‘osely‘bound stannous. To determine the total stannous (both tightly & loosely bound) present in a solution of stannous—associated complexes, a solution containing the complexes Was taken and placed into HN03 and incubated at room temperature with constant slow and overend mixing for 24 hrs. The mixture can then be centrifuged at about 10009, preferably for 15 minutes at room temperature. The supernatant when analysed for stannous ion content preferably by atomic absorption spectrophotometry (AAS), will provide the value for the total stannous t (whether that be boundlessociated with the x or free in solution). The level of loosetybound stanno-us in the solution can then be determined by taking a sample of a solution of stannous-associated complexes, placing it in a centrioon with a 1000 MWGO filter and centrifuging at about , preferably for 1, hour at room temperature, to produce enough te (<10% of total sample to not affect equilibrium) for analysis by AAS. The tes contain loosely—bound stennous ions. Tightly CPP~bound (colloidal r‘etentate). stannous ions can then be calculated from the ence between total and loosely— bound stennous ions. ‘Looser-bound’ etannous is stannous that is separable from a complex only by a method such as centrifugation as described above. The. loosely— bound stannous is still associated with the complexes but is more easily iable than tightly—bound stannous ions by the less stringent conditions explained above. 2E) In one embodiment, the us-associated stabilized amorphous calcium phosphate (ACP) or stannous—associated amorphous calcium fluoride ate (ACFP) complex including stannous that cannot be separated from the complex by centrifugation at about 30mg, preferably for 1 hour at room ature. in a further aspect of the present invention there is provided a method of mineralizing a dental surface sing providing (a) a composition including a stannous compound and stabilized ACP or ACFP! or (b) a stannousessooiated stabilized ACP or ACFP. in a preferred embodiment the dental surface is . in Va further aspect of the present invention there is provided a method for treating fluorosis comprising contacting a fiuorotic lesion in tooth enamel with (a) a composition including a stannous compound and stabilized ACP or AGFP, or (b) a stanno‘us- associated stabilized ACP or ACFP.

In a tﬂer aspect of the present invention there is provided a method for treating dental carie - mprising contacting a caries lesion in tooth enamel with (a) a composition [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r including a stannous compound and stabiiized ACP or ACFP, or (b) a stannous— ated stabilized ACF’ or ACFP. in a further aspect of the present invention there is provided a method for treating dentai erosion comprising contacting a lesion in tooth enamel caused by erosion with (a) a composition including a stannous compound and stabilized ACP or ACFP, or (b) a stannous-associated stabilized ACP or ACFP.

In a r aspect of the present invention there is provided a method for inhibiting the progression of dental erosion sing contacting a surface exhibiting erosion with (a): a composition ing a stannous compound and stabilized ACP or ACFP. or (b) a stan‘nous—associated stabilized ACP or ACFP, thereby forming a surface layer that inhibits the progression of dental erosion.

In a further aspect of the present invention there is ed a method for reducing dentinai hypersensitivity on exposed dentine comprising contacting the dentine with (a) a composition including a stannous compound and stabiiized ACP or ACFP. or (b) a stannous-associated ized ACP or ACFP. in a further aspect of the present invention there is ed a method for remineralizing a lesion in tooth enamel comprising contacting the iesion with (a) a composition inciuding a stannous compound and stabitized ACP or ACFP, or (b) a ue associated stabilized ACP or ACFP. 2O in a further aspect of the present inVention there is provided a method of increasing the reminera‘lization efficacy of a composition having stabiiized ACP‘ and/or ACFP the, method including the step of adding a stannous compound to the composition. ably, the stannous compound is added to the composition when it is in aqueous state, such as during manufacture.

Preferably the stannous—associated ized ACP or ACFP is stabilized by a phosphopeptide. in a preferred embodiment the phosphopeptide is a casein phosphopeptide. Preferably, the ACP or ACFF‘ is in the form of a casein phosphOpeptide stabilized ACP or ACFP‘ compiex. in any aspect or embodiments as described herein, the stabiiized ACP and/or ACFP may be in a formulation with additional calcium phosphate. Typically, the formulation amouay weight of m phosphate.inciud a PP stabilized ACP and/or ACFP compiex together with at least an equai [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by r [Annotation] wilksar Unmarked set by wilksar A ition including a stannous compound and stabiiized ACP or ACF‘P may be contacted with the dental surface for a period of about i to 60 minutes, or for about 1 to minutes. In one embodiment, the composition including a stannou-s compound and stabilized ACP or ACFP is contacted with the dental surface for about 20 minutes. An example of how this is achieved is formulating stannous compound. and stabilized ACP or ACFP into an oral ition, such as a paste, and then contacting. or applying the composition to. the dental e. The orai composition, such as a paste, has sufficient viscosity to be retained on the tooth for the required time period.

Preferably a composition including a s-tannous compound and stabilized ACP or ACFP ' is contacted with the dental surface for a period of about 1 minute to 2 hours, or 5 minutes to 60 s or about 10 minutes. The a composition including a stannous compound and stabilized ACP i’ ACFP may be repeatedly applied to the dental e over a period of 1 day to several months.

A us-associated stabilized ACP or ACFP may be contacted with the dentai surface for a period of about 1 to 60 minutes, or for about 1 to 30 minutes, in one embodiment, the stamens-associated ized ACP or ACFP is contacted with the dental surface for about 20 minutes. An exampie of how this is achieved is ating the stannous~associated stabiiized ACP or ACFP into an era! composition, such as a paste, and than contacting or applying the composition to the dental surface. The ore! 29 composition, such as a paste, has sufficient viscosity to be retained on the tooth for the required time period.

Preferably a stannous—associated ized ACP or ACFP is ted with the dentai surface for a period of about 1 minute to 2 hours, or 5 minutes to 80 minutes or about minutes. The stannous-essociated stabiiized ACP- or ACFP may be repeatedly applied to the dental surface over a period of 1 day to several months. ln one embodiment, the dental surface is in need of such treatment. Therefore, in another aspect, the invention includes in addition to the steps of any method described herein a step of identifying a subject suffering tiuorosis, dental caries, dentinai hypersensitivity or dental calculus, a white spot iesion; a fiucrotic lesion; a caries tesion; 3C) or a lesion caused by tooth erosion, dentai piaque, gingivitis or periodontitis.

The present invention es (a) a composition inciuding a us compound and stabilnl ACP or ACFP, or (b) a stannous-associated stabilized ACP or ACFP for use ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar in m‘ineralizing a dental surface or sob-surface and reducing the viabiiity of bacteria on that dental surface.

In any method of the invention, the stannous compound and stabilized ACP or ACFP are applied to the dental e sequentially or Concurrently. In any embodiment, the stan‘nous compound is added prior to the stabilized ACP or ACFP. In any embodiment, the stannous compound is added after the stabiiized ACP or ACFP.

A stanncus—associated stabilized ACP or ACFP compiex as referred to herein include a stannousassociated stabilized~ACP or ACFP complex. formed at a pH of beiow TO.

Preferably the complex is formed at a pH in the range of about 5.0 up to but below 7.0.

More preferably the complex is formed at a pH range of about 4.0 to 6.5, or 5.0 to about 6.0. In one embodiment, the pH during formation is maintained at pH 8.5 or below. in a preferred embodiment? the complex is formed at a pH of about 5.5. ably, the ACP or ACFP in the complex. is predominantly in a basic form. The stamens-associated stabilized ACP or ACFP complex when produced on an industriat scale is produced in a bulk solution that has a pH greater than about 7.0, preferably about 9.0; r the local pH at formation of the xes is below about TO, preferably about 4.0 to 6.5,, preferably about 5.5.

When staznnous-associated ized ACP or ACFP, or stabilized ACP or ACFPY is produced in the laboratory, in smaller quantities than commercial production the pH of 2O the entire solution may be maintained at a given pH, is if the CP was prepared at pH 5.5, then the entire solution during OPP-ASP formation was maintained at pH 5.5. r, it may be neither necessary nor desirable to reduce the pH of the entire bulk solution in commercial manufacture to 5.5 as the only part of the bulk solution required to have the acidic pH is where the complexes are forming and the bulk solution can have, and does have, localised fluctuations in pH. The pH fluctuations arise ularly from protons provided by the phosphate compound, for example dihydrogen ate, as it is added and the protons liberated from acidic phosphate ions when they convert into the basic form. P0431 Therefore, while the overall pH of the bulk on may be at above 7.0, for example about pH 9‘ the iocalised pH at which the OPP—ADP is formed is lower, typically below 10 or 85, preferably about 4.0 to 8.5, more preferably about 5.5.

These fluctuations are localised due to the size of the bulk on.

One ﬁned for forming a stamens—associated stabiiized ACP of the invention is a math omprising the steps of: [Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r ionNone set by wilksar [Annotation] r Unmarked set by wilksar (i) obtaining a solution comprising at toast one phosphopeptide and; (ii) admixing solutions comprising oaioium ions? phosphate ions and hydroxide ions, while maintaining the pH at about 7.0 or beiow; and (iii) admixing a stannous compound; (i) providing a solution of stabiiized ACP; and (ii) admixing a etannous compound.

One method for forming a ushassociated s-tabiiised ACFP is a method comprising the steps of: (i) obtaining a solution oomprieing at least one phosphopeptide and; (ii) admixing solutions sing oa‘ioium ions, phosphate ions, hydroxide ions and ﬂuoride ions, while maintaining the pH at about 7.0 or beiow; and (iii) admixing a st‘annous compound; (i) providing a solution of stabiiized ACFP; and (ii) admixing a stannous compound.

The hydroxide ions may be titrated into the solution to maintain the phosphopeptide solution at an essentially constant pH. The calcium and phosphate ions may be titrated into the phosphopeptide solution with ocmstant mixing and at a rate that avoids the formation of a m phosphate pteoipitete in the phosphopeptide solution.

A stannous—assooiated stabilized ACP‘ may be produced by a method sing the step of admixing P and a stannotis compound in an aqueous soiutionl while maintaining the pH at about 6.5 or below.

A stannous—aesooiated stabilized ACFP may be produced by a method sing the step of admixing CPP—ACFP and a stannoue compound in an aqueous solution! while maintaining the pH at about 6.5 or below.

A stannous—assooiated stabilized ACP may be ed by a method comprising the steps of; (i) obtaining a solution comprising CPP—ACP; and; [Annotation] wilksar None set by r [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar (ii) admixing a stennous compound, while ining the pi-l at about 8.5 or below.

A stannous ised ACFP may be produced by a method comprising the steps of: (i) obtaining a solution comprising CPP-ACFP; and; (ii) admixing a stannous compound, while maintaining the. pH at about 6.5 or below.

Preferably! the stannous compound is stannous fluoride. Optionally, the methods of producing stannous-associated stabilized ACP or stannous-associated stabilized ACFP further comprise admixing Sodium fluoride in step (ii).

Preferably the pH is ined with an acid, such as HCl.

Preferably the solution sing OPP-ACP or FP is prepared by adding CP‘P- ACP or CPP-ACFP to distilled or deionised water.

A stannous—associated stabilized amorphous calcium phosphate (ASP). andi’or amorphous calcium fluoride phosphate (ACFP) complex may" be formed by mixing Stabilized stabilized amorphous calcium phosphate (ACP) andl’or ous calcium fluoride phosphate (ACFP) complex with stannous fluoride.

A stabilized-ASP or ACFP complex as described in the current specification may be the d” complexes are shown in Figure 2 of Cross stat, .2007.

A stabilized-ACP or ACFP complex as referred to herein include a stabilized—ADP or ACFP' complex as described in PCTfAUZOOSI’OOWSl the contents of which are incorporated by reference. ln a preferred ment the phosphopeptide stabilised amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate ) complex has tightly bound and y bound calcium, wherein the bound calcium in the complex is less than the tightly bound calcium in an ASP or ACFP complex formed at a pH of 7.1:).

Optionally, the. ACP or ACFP is predominantly in a basic form.

A stabilized—ACP or ACFP complex as referred to herein include a stabilized-ACP or ACFP complex formed at a pH of below 7.0. Preferably the complex is formed at a pH in the range of about 5.0 up to but below 7.0. More preferably the complex is formed at a pH We of about 5.0 to about 6,0. in a preferred embodiment, the complex is formed [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] r None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar at a pH of about 55. Preferably. the ACP or ACFP in the complex is predominantly in a basic form.

A stabilized~ACP may be produced by a method comprising the steps of: (i) obtaining a solution comprising at least one phosphopeptide and; (ii) ng solutions comprising calcium ions, phosphate ions and hydroxide ions, while maintaining the pH at about 7.0 or below.

A stabilised ACFP may be produced by a method comprising the steps of: (i) ing a solution comprising at least one phosphopeptlde and; ( ii) admixing solutions comprising calcium ions. phosphate ions1 hydroxide ions and fluoride ions, while maintaining the pH at about 7.0 or below.

A phosphop-eptide stabilised amorphous calcium phosphate (AC-P) or amorphous calcium fluoride phosphate (ACFP) complex. may also include wherein the ACF’ in the complex has tightly bound and looseiy m: wherein the tightly bound, m in the complex is less than the tightly bound m in an ASP or ACFP complex formed at a pH of 7.0 and the ACP or ACFP is predominantly in a basic form, obtainable or obtained by a method comprising: a) admixing a first solution comprising calcium ions, at second solution comprising phosphate ions. and optionally a third solution comprising de ions, to a solution comprising phosphopeptides and a solvent with a. pH of from about 5 up to but below 7; and b) maintaining the pH of the solution at about 5.0 up to but below 7.0 during the ng by adding hydroxide ions.

“‘Tightiy" and “loosely? bound calcium and phosphate in ASP or ACFP can be determined using analytical ultrafiltration. Briefly, the solution of opeptide, calcium, phosphate and optionally fluoride admixed while ining the pH at about To or below can be first filtered through a 0.1 micron filter to remove free calcium andE phosphate that is not associated with the complexes. This free calcium and phosphate is present in the filtrate and discarded. Any free calcium or phosphate that is not associated in any way with the complexes» would not be bioavaiiable, is. delivered by the phosphopeptide to the tooth. The retentate from the 0.1 micron filtration can be nialyzed by centrifugation through a 3000 mw cutoff filter at 1,000 g for 15 min.

[Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar The ing filtrate contains calcium and phosphate that is loosely bound or associated with the complexes. At this centrifugal force calcium and phosphate that is not tightly bound to the complexes are released and move to into the filtrate. The Ca and Pi that is y bound in the complexes is retained in the retentate. The amount of tightly bound Ca and Pi in the retentate can then be determined by subtracting the amount of Ca and Pi in the filtrate from the total amount of Ca and Pi in the retentate of the 0.1 micron filtration.

A stabilized-ACP or ACFP complex as referred to herein include a stabilized-ACP or ACFP complex. as described in PCT/AUQOOSIOOOSSS the contents of which are ' incorporated by reference.

A “superloadect” phosphopeptide or phosphoproteln (PP) stabilized-amorphous calcium phosphate (ASP) or amorphous m fluoride phosphate (ACFP) complex. The complex may be formed at any pH (eg 3-13). Preferably the phosphopeptide includes the sequence -A—B-C-, where A is a phosphoamino acid, preferably phosphoserine, B is any amino acid including a phosphcamino acid and C is glutamlc acid, aspartic acid or a oamino acid. The phOSphoamino acid may be phosphoserine. The PP is superloaded with calcium and phosphate ions. The calcium ions may be in the range 0 mot Ca per mole of PP, or in the range of 30400 or 30450 mole Ca per mole of PP. in another embodiment, the mot Ca per mol of PP is at least 25, 30, 35, 40, 45 or 2t] 50.

The phosphopeptide or phosphoprotein (PP) stabilized amorphous calcium phosphate or amorphous calcium de phosphate complex may have a calcium ion content greater than about 30 moles of calcium per mole of PP. In a preferred embodiment, the m ion content is in the range of about 30 to too moles of calcium per mole of PP.

More preferably, the calcium ion content is in the range of about 30 to about 50 moles of calcium per mole of PP, The opeptide or pho’sphoprctein (PP) ized—amorphous calcium phosphate (ACP) or amorphous calcium fluoride phOSphate {ACFP} complex may be produced by a method sing the steps of: (i) obtaining solutions comprising calcium, inorganic phosphate and fluoride (optional); and (ii) admixing (i) with a on comprising PP-ACP.

[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by r [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar ln a preferred embodiment, the PP is casein phosphopeptide (CPP), The PP stabilized ACP and/or ACFP complex may further include at least an equal amount by weight of calcium ate. Preferably the calcium phosphate is CaHP04 Preferably, the calcium phDSphate (eg. CaHPO4} is dry blended with the PP stabilized ACP andfor ACFP complex. In a preferred embodiment, the PP—ACP andfor PP—ACFP complex: calcium phosphate ratio is about 1:1-50, more preferably about i: 125, more ably about 1:545. In one embodiment, the PvaACP andfor PP~ACFP complex: calcium ate ratio is about 1:10.

The oral care formulation that includes. a phosphopeptide or phosphoproteln (PP) Stabilized amorphous calcium phosphate (ACP) andfor amorphous calcium fluoride phosphate (ACFP) complex having a calcium ion content greater than about, 30 moles of calcium per mole of PP when used in the oral cavity may be produced. by a method ing the steps of: (i) obtaining a powder including a PP-ACP and/or PP-ACFP complex; (it) dry blending with an iVe amount of calcium phosphate; and (iii) formulating the dry d PP~ACP andlor PP-ACFP and calcium phosphate e into an oral care formulation.

Preferably, the form of calcium phosphate for dry ng is any e calcium phosphate including, but not limited to, CaHPO4, 4 and calcium lactate.

The present invention also provides a method of mineralizing a dental surface or sub- surface ing the steps of: (i) contacting the dental surface with a protein disrupting agent, and (ii) contacting the dental surface with {a) a composition including a stannous compound and stabilized ACP or ACFP, or (lo) a stannoosessociated stabilized ACP or ACFP, The dental surface is preferably dental enamel. ln one embodiment the dental surface is a lesion in the enamel, Such as a lesion caused by caries, dental erosion or fluorosis.

Any suitable protein disrupting agent can be used in the method of the. present invention. The agent is required to reduce the proteinaceoue barrier formed over the surface to be treated, suoh as the pellicle over teeth Examples of suitable agents inclucnbleach, detergent, chaotropi-c agents such as area, high phosphate [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar concentrations, cocktails of professes (eg. endopeptidases, proteinases and exopeptidases) and any other n solubilizing, disrupting or hydrolysing agent. es of suitable bleaohes include sodium hypochlorite (NaOCI), and d'e de bleaChes. ln a preferred ment, the bleach is an alkaline bleach. in a further preferred embodiment the alkaline bleach is NaOCl. The protein disrupting agent acts to solubilize and lly or wholly remove proteins from the dental surface, particularly ns of the pelliole.

A composition as described herein may further include free fluonde ions. The. fluoride ions may be from any suitable . A source of fluoride ions may include free ' fluoride ions or fluoride salts. Examples of sources of fluoride ions include, but are not limited to the following: sodium fluoride, sodium monofluorophosphate, stannous fluoride, sodium siliCOfluoride and amine fluoride. These may be provided in solution (typically an s solution), or a suspension.

The fluoride ions are preferably present in the composition in an amount greater than 1ppm. More preferably, the amount is more than 3 ppm. in another ment, it is preferably more than 10 ppm. in typical embodiments described below, the amount may be several hundred or thousand ppm. The fluoride content is lly measured as a ppm in oral compositions in the manner commonly used in the art. Where the fluoride is provided from a source with the stabilized .ACP, the ppm refers to the 2t] concentration of the fluoride in that source, typically a solution or suspension of oioavailable de.

“Phosphopeptide” in the context of the description of this invention means an amino acid sequence in which at least one amino acid is phosphoryiated. Preferably, the phosrihopeptide includes one or more of the amino acid sequence *AHBHC: where A is a phosphoamino residue, B is any amino acyl residue including a phosphoamino residue and G is ed from a giutamyi, aspartyl or phosphoamino residue. Any of the phosphoamino residues may independently be a phosphoseryi residue. B is desirably a residue the side—chain of which is neither relatively large nor hydrophobic. it may be Gly= Ala, Val, lit/leta Leu, lie= Ser, Thr, Cys, Asp, Glu, Asn, Gin or Lys. 3C) In another embodiment, at least two of the phosphoamin'o acids in the sequence are preferably uous. ably the phosphopeptide includes the sequence D- E. 'wﬁ A, B. C, D and E are independently phosphoserine, phosphothreonlne, ohos yrosine, ohistidine, giuta‘mio acid or aspartio acid, and at least two, [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar preferably three, of the A, B, C, D and E are a ohosphoamino acid. in a preferred embodiment, the phosphoamino acid residues are phosphoserine, most preferabiy three contiguous phosphoserine residues. It is also preferred that D and E are independently g'lutamic or aspartic acid. in one embodiment, the ACF’ or ACFP is stabiiized by a casein phosphopeptide (CPP), which is in the form of intact casein or fragment of the casein, and the x formed preferably has the a ACP}g]n or [(CPP)(ACFP}g]n where n is equal to or r than 1, for example ‘6. The. compie’x formed may be a. colloid-a! complex, where the core particles aggregate to form iarge (eg 100 nm) coiioidai particles suspended in ' water. Thus, the PP can be a casein protein or a phosphcpeptide.

The PP may be from any source; it may be present in the t of a iarger poiypeptide, including a full iength casein polypeptide, or it may be isctated by tryptic or other enzymatic or chemical digestion of casein, or other phosphoamino acid rich proteins such as phosphitin, or by chemicai or recombinant synthesis, provided that it comprises the sequence —A;-B-C— or A—B-C-D—E as described above. The sequence flanking this core sequence may be any sequence. However, those flanking ces in e31(59—79) [1], 33(1-25) [2], (132(46—70) [3] and Ctszi‘iﬁ‘i) [4] are red. The ﬂanking sequences may optionally be modified by deletion, addition or conservative tution of one or more residues. The amino acid composition and Sequence of the flanking region are not critical.

Examples of conservative substitutions are shown in Tebie ‘i beiow.

TABLE 1 Original Residue ary Conservative Preferred Conservative Substitution Substitution —Gln—ysHisPhe [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar Original Residue Exemplary Conservative Preferred Conservative Substitution Substitution Arg, Gin, Asn Leu, Vai, iie, Aia GIL: Trp Phe Thr' Ser The flanking sequences may also include non—naturaiiy occurring amino acid residues.

Ccmmoniy encauniered amino acids which are not enccded by the genetic code, inciude: o adipic acid (Aad) for Giu and Asp; E-aminopimelic acid {Apm) for Giu and Asp; E—amincbuiyric (Aim). acid for Met, Leu, and other aiiphat‘ic aminc acids; 2~aminaheptanoic acid (Ahe) for Met, Leu and cther aiiphatic aminc acids; 2—am‘inoiscbutyric acid (Aib) for Gly; cyclohexyiaianine (Che) , and Leu and lie; homoarginine. (Hear) for Arg and Lys; 2, 3diaminopropionic acid (Dpr) for Lys, Arg and His; N~ethyiglycine (EtGiy) for Gly, Pro, and Ala; iasparigine (EtAsn) for Asn, and Gin; yiiysine (Hy!) for L‘ys; ailchydroxyliysine (AHyI) for Lys; 3-(ann hydrcxyproline (SHyp, 4Hyp) for Pro, Set", and TN; [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r ation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar atloisoleueine (Atle) fer lle, Leu, and Val; o-amidinophenylelanine forAle; N-methylglycine (MeGlyi, saroesine) for th, Pro, Ale, N—methyliseleucine (Melt-e) for lie; Nemaline (Nva) for Met and other aliphatic amine acids; Norleucine (Nle) for Met and other aiiphetic amino acids; ine (Om) for Lys, Arg and His; Citrulline (Cit) and nine suifoxide (M80) fer Thr, Asn and Gln; N-methylphenylelanine (MePhe), trimethylphenyiaianine, haio (F, Cl, Br and t) 1O phenylatanine, triflourylphenylaianine, fer Phe. in one embodiment, the P? is one or more phosphopeptides ed from the group consisting of o51(59-79) [1], 5) {2], mews-70) {3] and (153(1-21) [4],: {1 1 MebGlu—Ala—GluﬁeﬂPHle~Ser(P)~—-Ser(P)—Ser(P)«Gtu—Glu-lie—VahPro—Asn— Ser(P)-Val—Glu—Gln«l_ys79 (sewers) Argﬁ—Glu-Leu«Glu«Glu~Leu—Asn—Vat—Pro-Gly-Gtu-|ie«Vat—Gtu~8er(P)-Leu—Ser(P)— —SemPieIu—Glueemte—Thr-Arg25 [in—25) {3] Asn45-Ale—Asn—Glu—Glu-Gtu—Tyr—Ser—iie-Gly-Ser(P)-Ser{P)-Ser(P)—Glu-Glu— Ser(P)-Ala-G|u—Vel—Ale—Thr-Giu-GIu-Val-Lysw 0652(46—703 {4] LysRAsn—Thr-Met-Glu-Hianal»Ser(P)»Ser{P)—Ser(P)~G tusGlu—Ser-i le-lie»Ser{P)~ Gln~Glu~Thr~Tyr—Lysm 0:530 ~21 ), ln another embodiment of the invention, the stannouse-essooieted stabilized ACP endfor sten‘nous-assooiated ized ACFP complex is inoerperetecl into oral compositions such as toothpaste. mouth washes or formuiations for the mouth to aid in the prevention and/"er treatment of dentai caries, teeth decay, dentat erosion or ftuorosis, at hypersensitivity, dental pteque= gingivitis or periodontitis, The oral compositions comprising an amount of stannous-assesiated steioiiizect ACP andz’or ACFP sufficient to form a layer on a dental surface, preferably, the tayer has a caictum : phosphate ratio equivaient to normal apatite, for exampie the ratie is about 2:1. The layer may contain an ennnt of caicium that is about 20 wt%. Prefer-abiy, the iayer may exhibit about an etemental analysis of any One of the elements, such as calcium, phesphate, fluoride, [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar carbon andior stannous, as shown in any one of Table 3 or 4. The stannoua-essociated stabilized ACP andior ACFP complexes may comprise 0.016096. by weight of the ition, ably 1.060%. For oral compositions, it is preferred that the amount of the stannoue-assooiated stabilized A-CP or ACFP complexes administered is 0.01 — 50% by weight, preferably 1.0% - 50% by weight of the composition. in a particularly preferred embodiment, the oral composition of the present ion contains about 2% stannous—associatad stabilized ACP or ACFP complexes or a mixture of both, The orat composition of this invention which contains the above~rnentioned agents may be prepared and used in various forms appiicable to the mouth such as dentifrice inciuding 1C) toothpastes, owders and liquid dentifrices, mouthwashes, mouthrinses, mouth Sprays, varnish, dental cement, troches, chewing gums, dental pastes, gingivai massage creams, gargle tablets, dairy products and other foodstuffs. The oral composition according to this invention may further include additional well known ingredients depending on the type and form of a particular orai composition. Certain compositions of the invention such as toothpastes, toothpowders and liquid rices, mouthwashes, mouthrinses and mouth sprays have reiatively low viscosity and have a ralizing effect without significant residence time in the oral .

In certain preferred forms of the invention an oral composition may be substantially liquid in character, such as a mouthwash, rinse or spray. In such a ation the 2E) vehicle is typically a water-alcohol e bly ing a humectant as described below, Generally, the weight ratio of water to aicohoi is in the range of from about 1:1 to about 20:1. The total amount of watenalcohot mixture in this type of preparation is typicaily in the range of from about 70 to about 999% by weight of the preparation. The aicohoi is typically ethanol or isopropanol. Ethanot is preferred. in other desirable forms of this invention, the composition may be ntially solid or pasty in character, such as toothpowder, a dent's! tablet or a toothpaste (dental cream} or gel dentifrice. The vehicte of such solid or pasty orat preparations generally contains dentally acceptahie polishing material. Exampies of poiishing materials are water— insoiuble sodium metaphosphate, potassium metaphosphete, tricalcium phosphate, dihydrated m phOSphate, anhydrous dicaicium phosphate, calcium osphate, magnesium orthophosphate, nesium phosphate, calcium carbonate, hydrated alumina, caicined alumina, aiuminium siiicate, zirconium silicate, silicanntonite, and mixtures thereof. Other suitable polishing material include the particulate thermosetting resin-s such as meiaminee, ic, and urea—formaldehydes, [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] r None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar and cross-linked polyepoxiides and polyesters. Preferred polishing materials include crystalline silica having particle sizes of up to about 5 microns, a mean particle size of up to about 1.1 microns, and a surface area of up to about 50,000 omzl'g., silica gel or colloidal silica, and complex amorphous alkali metal aluminosilicate.

When visually clear gels are employed, a ing agent of colloidal silica, such as those sold under the trademark SYLOID as Syloid 72 and Syloid 74 or under the trademark SANTOCEL as Santocel 100, alkali metal aluminosiiicate complexes are particularly useful since they have. refractive indices close to the refractive indices of gelling agent-liquid (including water andfor ent) systems commonly used in ' dentitrioes.

Many of the so—oalled ”water insoluble" polishing materials are anionic in character and also include small amounts of soluble al. Thus, insoluble sodium metaphosphate may be formed in any suitable manner, for example as rated by Therpe‘s Dictionary of d Chemistry, Volume 9, 4th Edition, pp. 510-511. The forms of insoluble sodium metaphosphate irnown as Madrell‘s salt and Kurrol‘s salt are further examples of suitable materials. These metaphosphate salts exhibit only a minute solubility in water, and therefore are commonly referred to as insoluble metaphosphetes (lMP). There is present therein a minor amount of soluble phosphate material as impurities, usually a few percent such as up to 4% by . The amount of soluble phosphate al, 29 which is believed to include a soluble sodium trimetaphosphate in the case of insoluble metaphosphate, may be reduced or eliminated by washing with water if desired. The insoluble alkali metal metaphosphate is lly employed in powder form of a particle size such that no more than 1% of the material is larger than 37 microns.

The polishing materiel is generally present in the solid or pasty compositions in weight concentrations of about 10% to about 99%. Preferably, it is present in amounts from about 10% to about 75% in toothpaste, and from about 70% to about 99% in toothpowder. In toothpastes, when the ing material is silioious in nature, it is generally present in an amount of about ill—80% by weight. Other polishing materials are typically t in amount of about 3.045% by weight. 3C) in a aste, the liquid vehicle may comprise water and ant typically in an amount ranging from about 10% to about 80% by weight of the preparation. Glycerine, propyg glycol, ol and polypropylene glycol ify suitable hume . tel/carriers. Also advantageous are liquid mixtures of water, glyceri‘ne and [Annotation] wilksar None set by r [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar sorb‘itcl. in clear gels. where the refractive index is an important consideration, about 2.5 ~ 30% wfw of water, 0 to about 70% wiw of glycerine and about 206094} w/w of sorbitol are ably ed, Toothpaste, Creams and gels typically contain a natural or synthetic thickener or gelling agent in proportions of about 0.1 to about 10, preferably about 0.5 to about 5% wlw. A suitable thickener is synthetic hectorite, a synthetic colloidal magnesium alkali metal silicate complex clay ble for example as Laponite (eg. CP, SP 2002, D) marketed by Leoorte Industries Limited. Laponite D is, imately by weight 58.00% SiOz, .40% MgO, 3.05% N820, 0.98% LiZO, and some water and trace metals. its true ' specific gravity is 2.53 and it has an. apparent bulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include lrish moss, iota carrageensn, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl methyl ose, hydroxyethyl cellulose. (eg. available as Natrosol), sodium carboxymethyl cellulose, and colloidal silica such as finely ground: Syloid (eg. 244). Solubilizing agents may also be included such as humectan‘t poiyols such propylene glycol, dipro-pylene glycol and hexylene glycol, ce‘llosolves such as methyl celloSolve and ethyl cellosozlve, vegetable oils and waxes containing at least about 12 carbons in a straight chain such as olive oil, castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate. it will be understood that, as is conventional, the oral preparations will usually be sold. or otherwise distributed in suitable labelled packages. Thus, a jar of mouth rinse will have a label describing it, in substance, as a mouth rinse or mouthwash and having directions for its use; and a toothpaste, cream or gel will uSually be in a collapsible tube, typically aluminium, lined lead or plastic, or other squeeze, pump or pressurized dispenser for metering out the ts, having a label bing it, in substance, as a toothpaste, gel or dental cream.

Organic surface—active agents may be used in the compositions of the present invention to achieve increased prophylactic action, assist in achieving thorough and complete sion of the active agent throughout the oral cavity, and render the instant 3C) compositions more cosmetically acceptable. The organic surface-active material is preferably anionic, non—ionic or ytic in nature and preferably does not interact with ﬁctive agent. It is preferred to employ as the surface-active agent a, detersive mate - hich imparts to the composition detersive and foaming properties. Suitable ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar examples of anionic tants are water-soiuble salts of higher fatty acid monoglyceride monosulfates, such as the sodium sail of the monosulfated monoglyoeride of hydrogenated coconut oii fatty acids, higher alkyl sulfates such as sodium lauryl sulfate, alkyl and sulfonates such as sodium dodecyl e sulfonate, higher alkyisulfo-acetates, higher fatty acid esters of 1,.2-dihydroxy propane sulfonate, and the substantially saturated higher aliphatic acyl amides of lower aliphatic amino carboxyiic acid compounds, such as those having 12 to 18 carbons in the fatty acid, alkyl or acyl radicals, and the like. Examples of the last mentioned amides are N—lauroyl sarcosine, and the sodium, potassium, and ethanolamine salts of N~lauroyl, Namyristoyt, 1t] or N—palrnitoyl sarcosine which should be substantially free from soap or similar higher fatty acid material. The use of these sarconite nds in the oral itions of the present invention is particularly advantageous since these materials exhibit a- prolonged marked effect in the inhibition of acid formation in the oral cavity due to carbohydrates breakdown in addition to ng some reduction in the solubility of tooth enamel in acid solutions. es of water-soluble non-ionic surfactants suitable for use are condensation products of ethylene oxide with various reactive hydrogen— containing compounds reactive therewith having tong hydrophobic chains (eg. aliphatic chains of about 12 to 20 carbon atoms), which condensation ts xamers"); contain hydrophilic polyoxyethylene moieties, such as condensation ts of poly (ethylene oxide) with fatty acids, fatty alcohois, fatty amides, poiyhydric alcohols (eg. sorbitazn monostea'rate) and polypropyleneoxide (eg. Pluronic materials).

The surface active agent is typically present in amount of about O.‘l—5% by . it is noteworthy, that the e active agent may assist in the dissolving of the active agent of the invention and y diminish the amount of solubilizi‘ng humeotant needed.

Various other materials may be incorporated in the oral preparations of this invention such as whitening agents, preservatives, silicones, chlorophyll compounds andfor ammoniated material such as urea, diammonium phosphate, and mixtures thereof.

These adjuvants, where present, are incorporated in the preparations in amounts which do not substantially adversely affect the properties and characteristics desired.

Any suitable" flavouring or sweetening material may also be employed. Examples of suitable ring constituents are devouring oils, eg. oil of int, peppermint, Wintergreen, sassafras, clove, sage, eucalyptus, marioram, cinnamon, lemon, and oranﬂnd methyl salicylate, Suitable sweetening agents include sucrose, iactose, [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r maltose, sorbitoi, xylitoi, sodium ate, perillartlne, AMP (aspartyl phenyl alanine, methyl ester), saccharine, and the like. Suitably, flavour and sweetening agents may each or together comprise from about 0.1% to 5% more of the preparation.

The itions of this invention can also be incorporated in lozenges», or in chewing gum or other products, eg. by ng into a warm gum base or coating the outer surface of a gum base, illustrative of which are islutong, rubber latex, vinylite resins, etc, desirably with conventional cizers or softeners, sugar or other sweeteners or such as glucose, sorbitol and the like. The composition of the invention may be a dual phase ition wherein each phase permits release of components over different ' time periods. For example, in use a dual phase composition may release stannous» associated stabilized ACP andi’or stannoussassooiated stabilized ACFP, ably CPP-ACPfSan andior CPP—ACFPlSan, from a first phase at a faster rate than a compound that is capable of sing or maintaining the pH of a solution from a second phase. Preferably, the dual phase composition is a dual phase chewing gum.

An alternative composition may be one that es stabilized ACP or ACFP and a us compound that then in situ, such as the oral cavity, forms stannous- ated stabilize-d ACP or ACFP. An exemplary composition may be a chewing gum that contains ized ACP or ACFP in the pellet and a stannous compound in the centre chew. 2O in a further aspect, the invention provides compositions including pharmaceutical compositions comprising any of the (a) compositions including a stannous nd and stabilized ACP or ACFP, or (is) a stannoussassociatecl stabilized ACP or ACFP xes as described above together with a compound capable of increasing or maintaining the pH of a solution and a ceuticallyaacoeptaole carrier. Such compositions may be selected from the group consisting of dental, antioariogenic compositions and therapeutic compositions. Dental compositions or therapeutic compositions may be in the form of a gel, liquid, solid, powder, cream or lozenge.

Therapeutic compositions may also be in the form of tablets or capsules. In one embodiment, the stamens—associated stabilized ACP or ACFP complexes are ntially the only remineralizing active components of such a composition. For example, a creme formulation may be employed containing: water; glycerol; OPP- A-CPfSnFZ; D—soroitol; silicon dioxide; sodium oarboxymethylcellulose (CIVIC-Na); propyue glycol; titanium dioxide; xylitol; phosphoric acid; guar gum; zinc oxide; sodium [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar saccharin; ethyl p~hydroxybenzoete magnesium oxide; butyi oxybenzoete and propyi p~hydroxybenzoate The invention r includes a formulation described above provided together with instructions for its use to treat or t any one or more of dental Caries or tooth decay, dental erosion and fluorosis, dentinal hypersensitivity, dental plaque, gingivitis or periodontitis.

In another embodiment, the compositions of the ion as described herein do not e a ate buffer andlor a calcium cheiator. For example, any dentifrice described herein may not include a ate buffer andior a calcium or. in an embodiment of the present invention there is provided a composition for dental mineralization including stannoue—associated stabilized ACP or ACFP complexes, wherein the composition does not e a phosphate buffer endior calcium chelatort in another embodiment, the compositions of the invention as described herein do not inci‘ude a viscosity regulator: or a viscosity regulator at 0.5 to 50%. in another embodiment, the compositions of the ion as described herein do not include sodium yme‘thylceiiuiose, or 0.01 to 10% sodium carboxymethylceliuloee having the esterification degree of 0.7 to it). in one embodiment, the active components of the composition consist essentiaily of the stennous-associated stabilized, ACP or ACFP complexes.

In a further aspect, there is provided a method of treating or preventing one or more of each of dental caries. tooth decay, dentai erosion and fluorosis, dentinel hypersensitivity, dental plaque, gingivitis and periodontitis comprising the steps of administering (a) a composition including a stennous nd and stabilized ACP or ACFP, or (o) a stamens—associated stabilized ACP or ACFP‘ oompiexes, to the teeth of a subject. Topical stration of the complex is preferred. The method preterabiy includes the administration of the complex in reformulation as described above.

In a further aspect there is provided the use of (a) compositions including a stannous compound and stabilized ACP or AC-FP, or (b) a stennous-eseociated ized NIP or ACFP complexes in a manufacture of a composition for the treatment and/or prevention of one or mere of dental caries tooth decay, dental erosion and tluorosis, dental hyperusitivity, dental plaque, gingivitis and periodontitis.

[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar Aocording to a further aspect of the invention there is provided a ition for dental restoration, including a dental restorative material to which has been added (a) a ition including a us compound and stabilized ACP or ACFP, or (b) a, stamens—associated stabilized ACP or ACFP complexes. The base of the dental restorative al can be a glass ionomer cement, a composite al or any other restorative material which is compatible. A. glass ionomer cement is preferred. it is preferred that the amount of stabilised stannous‘aeeooiated stabilized ACP or ACFP complexes, preferably CPP-AGPfSan complex or CPP—ACFP/San complex, included in the dental ative material is 0.0‘l~80% by weight, preferably 0.540% and more 1C) preferably 16% by weight. The dental ative material of this invention which: contains the above mentioned agents may be ed and used in various forms applicable to dental practice. The dental restorative material according to this embodiment may further include other ions, eg. antibacterial ions Zn”, Agi etc or other additionai ingredients ing on the type and form of a particular dental restorative materiai. It is preferable that the pH of dental restorative material according to this embodiment be between 240, more preferably 59 and even more preferably 5-7. it is preferable that the pH of the dental ative material containing a stannoue associated stabilized ACP or ACFP complex be in the range of about 2 to 10‘ more preferably in the range of about 5 to 9 and even more preferably in the range of about 5 to 7. it will be y understood that, although this specification refers specificaity to applications in humans: the invention is also useful for veterinary purposes. Thus in all s the invention is usefut for domestic animals such as cattle, sheep: horses and poultry; for oompanion animals such as oats and dogs; and for zoo animals.

One example of a mineralizing composition comprises the following (in decreasing. order of proportion}: water glycerol CPP-ACPISan D-sorbitoi silicon dioxide sodium carboxymethyloellulose {CIVIC-Na) ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar propylene glycol titanium e xy‘iitoi phesphoric acid guar gum zinc oxide sodium saccharin ethyi oxybenzpe‘te magnesium oxide butyi p~hydroxybenzoate propyl pwhydroxybenzoete The invention wiil now be further described with reference it) the following non~limiting examples.

Example 1 Casein phosphopeptide-amorphous calcium phosphate (OPRACP) was acquired from ry Enterprises Pte Ltd under the natk name Recaident-TM. A solution was. prepared using CPP~ACP, 8an and NeF te produce at 0.4% w/v CPP-ACP, 220 ppm F as :3an and 7’0 ppm F as NaF, pH 5.6. Speciﬁcally, the CPP~ACPISan complexes were prepared by adding CPP-ACP to distilled/deionised water and then SHE-2 (solid) and Net: added with addition of 'i M HCi to maintain the pH between 4.0 — 6.5; The pH was not allowed to go above 65. The total volume of acid added was less then We of the PISan solution volume. This solution was designated stabilized Sanz’ACF’.

While NaF was added in this method it is a minor component and the majority of the flupride derives from the San. The method pduid be performed using $3an- only {without NeF). Another selution was prepared using CPP—ACP and NeF to produce 0.4% wfv CPP—ACP, and 290 ppm F as NaF, pH 5.6. This solution was designated stabilized Na'FIACP.

Both solutions were stable at roam temperature (20°C) for many months with no Both solutions were tested for their ability to remineralize enamel precidﬂesubs 'e lesions. Human teeth enamel reiized subsurface lesions were [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r prepared in third melar enamei biocks using the method of Reynolds, (1997’). J Dent Res 716215874595. Half the blocks were remineraiized by suspending them individually in the stabilized SanlACP solution and the other half in the stabiitzed NaFlACP solution for 7 days at 37°C. After remineraiization the enamel biocks were embedded, sectioned and subjected to transverse microradiography and densitometric image analysis as previously described by Reynolds (199?) to determine percent mineral content gain (%Remineralization) shown in Tabie 2. A one way analysis of variance with differences in means determined using a Tokay HSD post hoc comparison showed that the treatment with ized SanlACP solution significantly increased the level of remineralization by 32%.

TABLE 2. Remineralization of enamel subsurface lesions by stabilized SanlACP versus stabilized NaFfACP.

Solution ﬁt: Remineralization Stabilized SnF2/ACP {0.4%w1’v CPP—ACP 220 ppm F as 8an 70 ppm F as NaF] Stabilized P {0.4% ‘w/v CPP—ACP 290 ppm F as NaF] *signiﬁcantly different p < 0.01 Analysis of the enamel lesions using scanning etectron microscopy (Figures 1 and 2) revealed that the lesions d with the stabilized Sani’ACP r had a surface layer. Surprisingly the stabilized P formuiation produced icantly greater enamel subsurface lesion raiization (Tabte 2) and appeared also to form a protective surface iayer demonstrating the cted superiority of the stabilized P formulation. An experiment using scanning electron microscopy —- energy dispersive spectroscopy EDS) of the surface iayer (Figure 3 and Table 3) confirmed that it was apatite. The surface layer EDS is showed it contained .3 % P and 21.18 wt% Ca giving a typical apatite Ca:P wt% ratio of 2.35. The [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r sound enamel contained 15.28 wt% P and 30.99 wt% Ca giving the same typicat apatite CazP wt% ratio of 2.03.

These data show that the surface layer is composed of apatite, however the surface layer had higher Sn (1.23 ‘wtﬂ/B versus 0 wt% for sound ) and fluoride (0.54 wt% versus 0.11% for sound ). This of course is consistent with the (SPF-stabilised P solution containing Sn and F. OPP was also involved in the formation of the» surface layer as the C was 26.53 wt% in the surface layer but only around 10 wt% in. the sound enamel (Table 4); This supports the mechanism whereby the us ions can cross-tink the phosphop‘eptide stabilized ACP‘ or stabilized .ACFP at the tooth surfaoe to ' form a layer that can protect the tooth surface from dentineralization.

These s indicate that the SPF-stabilised SanlAGP treatment has not eniy produced substantially better subsurface remineratisation but it has also produced a uniform, protective surface layer.

The. retention of stannoue-associated stabilized ACP or ACFP in a cross—linked surface layer would (i) protect from tooth sensitivity as it would seal dentinal tubules, (it) protect from plaque formation as it contains the cterial ion Sn and (iii) protect from dentat erosion and dental caries. It woutd also e subsurface remineralization. Without being bound by any theory or mechanism of action it appears that. stannous ions help to promote the Surface tayer by cross—linking the CPP—ACP or CPP-ACFP at the surface of 2O the enamel.

TABLE 3. Etemental analysis by SEM-EDS of the mineratised surface layer resulting from treatment with stabilized SanlACP.

Normal Sound Enamel (WP/o) [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar A more detailed scanning electron copy ~ energy dispersive spectroscopy (SEM- EDS) analysis of the various layers wherein the embedding resin is excluded is shown in Table 4. This shows that the high carbon t in the sound enamel in Table 3 is. including the resin. When the resin was excluded, the carbon content of sound enamel was in the range of 6 to lOthxla. The carbon t of the surface layer produced by the CPP~stabilised P treatment was almost 8~foid greater than sound enamel indicating that the phosphopeptides are present in the layer" The tive surface layer formed by the stannous—assooiated stabilized ACP complexes provides a source of fluoride as the level of fluoride in the mineralised surface layer is 5 fold greater than the level of fluoride in sound enamel. Further, the level of fluoride t is. greater than that present in the remineralized lesion. The level of stannous present in the surface layer was significantly higher, 1.2 fold, than the remineralizecl lesion.

The level of fluoride in the lesion remiheralized with the stannous associated ACP is r than CPP-ACP and sodium fluoride. Further, there was an increase in calcium present in the lesion remineralized by the stahnous~aesociated stabilized ACP compared to the CPP-ACP and sodium fluoride. This level of calcium is approaching. the level of calcium present in sound enamel.

TABLE 4. ed elemental analysis by SEM—EDS of the mineralised, e layer resulting from treatment with OPP stabilized SanlA-CP.

Element Sound Demineralised CPP-ACPJNaF CPP— CPI”- wt% Enamel Remineralised A'CPi‘S'n F2 ACP/Sn F2 Lesion Fie— Mineralised mlneralised Surface Lesion Layer 40.59 i 0.63 38.14 i 0.32 37.94 $10.44 37.93 i 045; 39.59. i 0.238 [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar 0.12i0‘12 0.21:0.16 0.34i023 0.54i0.’19 (3.1 told (5. fold increase) increase) 1526:026 1364:013 1405:8748 t4.02i0.19' 1033:0113 .99 + o 44 27’9”? i 0.21 28.49 i 0.29 29.25 i 0.33 21.18 i 0.21 0.1 (11110.17 02 t.2-3:t0.16 (2.4 told (12.3 foid increase) increase) 2.031” 21151 2.086 2.050 a. Babel et al. Scientific World Journal (2012) t). ND = not yet determined {3. CezP wt% ratio for HA = 2,157 and for ACP = 1.941 Example 2 This exempie describes the. mental! protocoi for the measurement. of CPP~beunci (tightly bound), loosely bound and free ions in solution A sempie of each solution prep-erect in Exampie 1 was taken and less than 10% collected as e te using a 3000 moleouier weight f Centriprep 3 ultraﬁltration membrane. The Centripreps containing the samples were centrifuged at 1,000 g for 15 min in a Beckman J.2-21 centrifuge using a JA 10.5 rotor; The original sample before Centriprep centrifugation and a sample of the filtrate after Centriprep centrifugation were taken for analysis of calcium, phosphate fluoride and stennous concentrations. The is of the al sample gave total caicium, phosphate, fluoride and s-tannous ion concentrations and the analysis of the te gave free (unbound) calcium, phosphate and e concentrations. The difference between the total and unbound concentrations is the bound concentration of Ca, Pi, F and Sn by the EFF.

Example 3 A topn creme may be produced in accordance with the t invention having the following ingredients: ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar Water glycerol Sta‘biiised SanlACP and/or San/ACFP D—sorloitol sodium oarboxymethylcelluiose (CMC—Na) propylene glycol silicon dioxide titanium dioxide xylitol phosphoric acid sodium fluoride sodium rin ethyl p~hydroxybenzoate . propyl p-hydroxybezoate butyl p-hydroxybenzoata Example 4 A mouthrinse formulation may be produced in ance with the present invention having the following composition: Water Poloxamer 407 Sodium Lauryl Sutphate Stab'ilised S‘anlACP andjosr SanlACFP Sodium Fluoride Flav‘ours Sodiugaocharin [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar Ethyl p—hydroxybenzoate Proipyl oxybenzoete Butyl p-hydroxyoenzoate Example 5 A sugar—free chewing gum formulation may be produced in accordance with the present invention having the following composition: Crystalline sorbitola'mannitolfxylitol Gum base Calcium carbonate Glycerine Stabilised SanfACP andfor SaniACFP Sodium Fluoride Flavour oil Water Example 6 The following is a protocol for OPP stabilized ACP/San solution ion analysis. Total (tightly & loosely-bound) and loosely—bound samples were prepared as follows: Total ly and loosely—bound): One ml of 0.4% PXEQOppm F(as SnFQ/TOppm F(as NaF) solution was taken and placed into 19 ml of "ill/l HNOS and incubated at room temperature with constant slow and over and mixing for 24 hrs (20 rpm). The mixture was centrifuged at 10009 for 15 s at room temperature. The supernatant was analyzed for calcium, stannous, phosphate and fluoride.

Loosely-bound: A sample of the 044% CPP—ACPIEZOppm F(as SthWOppm F(as NaF) solution was. taken and placed in a centricon with a 10130 MWCO filter and centrifuged at '3000g for thour at room temperature to e enough te ( <‘10% of total sample to not affect equilibrium) for analysis by atomic absorption spectrophotometry (AAS) and ion chromatography (ill). The tes were then ed to give loosely— bound ions, [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar ed set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar The total and loosely bound calcium. stennous, phosphate and fluoride in the solution were determined by ion tography (for e and phosphate) and Atomic Absorption Spectrometry (for calcium and stannous).

CPP tightly-bound (colloidal retentate) ions were calculated from the difference between Total and y—bound (as explained above). Based on this example, the stannous— associated ACP complex had a stennous ion content of about 6 to .8 mole per mole of phosphopeptide.

Table 5. ion is of O.4%CPP~ACP!220ppm F as Oppm F as NaF solution at pH 5.5 and pH 4.0 by Atomic Absorption Spectrometry (for stannous and calcium) and 1O ion Chromatography (for fluoride and ate).

Il-16113005831 8138012153855) 5582251403 292.5-h’l. 7 Total 40 1704595924 957oo+1594 5572151159 28955120 . 15589 (95.2%) 9559 (91.7% 5545(99.9%)1202 (41.1%) Tightly- b0“““13759(59.9%) 5499953999) 515959.995) 1542(557%) 770-9 i 5.5 592.0 i 193.5 39.1 i 3.4 192.3 i 2.9 .5. (4.9%) (9.3%) (9.9%) (55.9%) bound 3257.1 i164 3170.7 5 55.1 522.3 919.11 125.9 $1.1 4 0' (19.1 19) (35.5%) (9.2%) (49.9%) This in situ study was designed to compare reminerelisation using an established intra- orel reminerelisetion model promoted by five solutions: : 1) 0.4% (w/v’) calcium phosphopeptide stabilised—amorphous, calcium phosphate ('CPP— ACP), 220 ppm -us fluoride (San) and 70 ppm sodium fluoride (NaF) (shown in Figure 7’ as 2%CPP«ACP+SnF2); 2) ﬂaw/v} CPP—ACP and 290 ppm NaF; [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar 3) 0.4% (will) OPP-ADP; 4) 220 ppm 8an and 70 ppm NaF without OPP—ASP (shown in Figure 7’ as San); and ') 290 ppm NaF. in this model subjects wear a palatal appliance and rinse 4 times a day with 5 ml. of solution for five 14 consecutive-day treatment periods (inoluding ds) and rinsing with a different solution during each treatment period.

This randomised controlled study used a double~blind, five~way crossover design to assess the effects of five ons to enhance enamel remineralisation using an intra- oral ralisation model. Each solution contained the same amount of 290 ppm F alent to 1450 ppm F in e toothpaste diluted t in 5) [see above]. Subjects were randomly ted to one of the five solutions for the first treatment period and, after a one-week t rest , crossed over to another on for the second treatment period. This was repeated for the five treatments. Each subject were a custom made palatal appliance containing four pro—sterilised enamel slabs containing artificially~oreated subsurface lesions and four times a day for 14 consecutive days including weekends (treatment period) rinsed for one minute with 5 mL of their allocated solution then expectorated all the solution and accumulated saliva and continued to wear their appliance. The four rinses per day were performed it) after breakfast, (ii) after lunch} (iii) after dinner and (iv) before retiring at night. After the first rinse per day of each treatment period subjects on three different days expeotorated the solution and accumulated saliva into a clean tube for ion analysis. Subjects kept a diary of times and duration they rinsed with their solutions. Subjects were instructed to in their normal diet and oral hygiene procedures for the duration of the treatment periods.

Appliances were removed during subjects normal oral hygiene procedures during the study period. After removing the appliances for oral hygiene procedures, subjects Cleaned their appliances as instructed with a toothbrush and fluoride—free denture paste (both supplied) avoiding the trough areas and gently rinsed their appliances with DDW before replacing the appliances. When out of the mouth the. appliances were stored in sealed humid containers. All subjects brushed their teeth with standard fluoride 3C) toothpaste ed by the sponsor for the on of the study. The ts returned to the al site with their appliances and diary at the conclusion of each 14-day treatrrﬁt period.

[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar The researchers did not know which treatment had been administered nor the. subject.

Neither the chers, nor the er had access to the treatment code. Personnel dispensing the test solutions or supervising their use did not ipate in the examination of the enamel half-slabs in order to minimize ial bias. Following the baseline ation, subjects were ed a subject number. t numbers were recorded on their case report forms. Subjects were randomly assigned to. use one of the solutions during the first ent period then crossed over to another solution for the second to fifth treatment period. Randomisation was effected using a rd randomisation table for the number of treatments in the crossover study. Results of the 1C) in situ study are shown in Figure 7. These results show that in an in situ model a Solution including stannous-associated stabilized complexes (as 2% CPP-ACP, with stannous fluoride and sodium ﬂuoride, far right column. aiso referred to herein as stabilized SnFafACP) provide a significantly greater level of enamel subsurface remineralization compared to sodium fluoride alone, stannous fluoride & sodium fluoride, CPP-ACP alone or CPP—ACP with sodium ﬂuoride. The two far right columns ed 2% CPP-ACP+NaF and 2% CPP-ACP+SnF2 were significantly different {p<0.01). This clearly demonstrates that in vivo in the oral cavity the stannous associated stabilized xes of the ion have a superior capacity to remineralize lesions. 2E) A palatal appliance for each subject was ed by taking alginate impressions of upper and lower dental arches from which study models were produced and articulated.

Removable palatal acrylic appliances covering the first premolars to the last tooth in the arch were fabricated for each subject. The appiiance consisted of a palatal plate that was retained in the mouth by four stainless steel Clasps. Troughs on each side of the appliance adjacent to the paiatal surface of maxillary premolarimolar teeth accommodated two enamel half—slabs containing ralised subsurface lesions.

Therefore, each appliance contained four enamel half—slabs. The appliances was designed to ensure its surfaces were smooth and comfortable for the subjects.

Extracted third molars were obtained from the Royal Dental Hospital of Melbourne and oral surgeons and general practitioners in private practice. Any ed soft. tissues was removed and the teeth washed in distilled zed water (DEW). All teeth in this study were sterilised by storage in a 10% (WV) neutral buffered formalin solution for at least D3 weeks at room temperature. After storage in formalin the teeth were thoroughly washed in DDW and stored in DDW until required. Sound relatively planar [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by r ouccal and l surfaces of sterilised enamel with minimal cracking, staining. and fluorosis (as viewed under a dissecting microscope) were selected and thoroughly rinsed with DDW again The outer enamel surface including superficial cracks was d then ed wet to a mirror finish using Softex"M (3M) polishing discs on a slow speed contra~angie dental handplece. Each polished surface was then sewn from the tooth as an approximately 8 x 4 mm slab, using a water—cooled diamond blade saw and the whole slab covared with esistant nail varnish except for two. (occlusal and glngival) mesiodistal windows (approximately ’l x 7 mm) separated from each other by about 1 mm. Lesions were created in the enamel windows by mounting each slab onto the end of a 3 - 4 cm stick of yellow dental sticky wax and immersing in 40 mL of unagitated demineralisation buffer, consisting of 80 leL Noverite K—702 (pol-ycacrylate, Lubri'zol Corporation, ffe, OH; White, 1987), 500 .mgrL hydroxyapatite ('Blo—Gel®v HTP, Bio Rad Laboratories, Richmond CL), and 0.1 molll. lactic acid (Ajax Chemicals, Auburn MSW} pH 48., for four days at 37°C. A change of solution was; made after two days at which time the slabs were removed from the solution, rinsed thrice with DOW, blotted dry and placed into fresh demineralisation butter. The slabs were similarly rinsed and dried after four days of demlneralisatlon. This demineralisation procedure produces consistent subsurface s 80 to 110' pm deep with intact surface layers, as evaluated by microradiography of sections of the artificial lesions. After demineralisation, each enamel slab was sewn through the midline of each window into two 4 x 4 mm slabs and the cut surface of- each slab covered with nail h. One half of each slab was retained as the demlneralisation control (control halt-slab) and stored in a labeled 0.5 mL entrifuge tube together with a drop of DDW, thereby creating a humidified environment. The other half of the enamel slab was inset into the intraoral appliance using dental wax for the reminerailsatlon protocol (test half—slab). Care was taken not to cover the artificial lesions with wax. At the end of an in situ treatment period the enamel half—slabs were removed and replaced with new pro—sterilised enamel half~ slabs for the beginning of a new test period, After each treatment period each test half- slab was paired with its control half—slab and embedded, sectioned and ed to detennln‘e mineral changes Each pair of enamel labs (remineralisation half-slab paired with its demineraiisation control half—slab had the nail varnish carefully removed and was rinsed thoro ly with distilled deioinlsed water (DOW) The pair of enamel half—slabs (test remineralisation half-slab paired with its ralisation control half—slab) was placed [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar onto their cut sides into a small plastic vial with the lesion windows parallel and transparent cold curing methacrylate resin (Palad'ur, Heraeus Kulzer, Germany) poured over the half—slabs and d to set at room temperature; In order to identify the test and control half—slabs, a permanent identifying mark was drawn on the side of the methacrylate block. next to the test enamel half—slant Sections 200-300 cm thick were cut from the embedded labs, perpendicular to the lesion surface through the midline of both half-lesions using an internal annulus saw microtome (Leitz 1600, Ernst Leitz Wetzlar, Germany). The sections were then lapped down to 80 :t 5 pm using a RotoPolnEl/Rotoi—“orceél g instrument (Struers, Denmark) with 1200 grit lapping paper, The lapped sections were removed from the lapping instrument and rinsed in DDW} d dry and stored on soft tissue between glass slides Each n, which contains the remineralised halt-lesion and the paired demineralised control half lesion from the same enamel slab, were radiographed along with an aiuminlum stepwedge of x 37.5 pm thick increments using Microchrome High Resolution glass plates (3” x 3” x 0.06"; Microchrome, USA) and nickel filtered copper Kn radiation at 20 kV and 30 mA for 8 minutes using an XMR microradiography system (Diffraction Technologies Pty Ltd) with a PANalyticai fine focus glass XRD tube with a copper target (PW2213/20» powered by a DFB generator (Spellman High Voltage Electronics Corporation). Each glass plate was developed in Microchrome per {)5 (1:4 dilution, Microchrome, USA) for five minutes, placed into glacial acetic acid stop bath for 30 seconds and fixed in Microchrome Developer F4 (1:4 dilution, Microchrome, USA) for five minutes. The ature of all the photochemicals was maintained at 20°C.

Radiographic images of the lesions were viewed via transmitted light through a Dialux microscope (Ernst Leitz Wetzlar, Germany). The images were acquired by a digital camera (lnsight'i‘t‘) under the l of imaging re (lmage Pro Plus version r)- running on a, PC (Pentium ,lll). images at the s and the neighbouring areas of sound enamel were scanned using the program’s line luminance function that gives readings in gray values between 0 and 256. An area free of artifacts or cracks was selected for analysis. Each scan comprised 200 readings taken from the tooth surface to sound enamel. The stepwedge image on each slide was d and the averaged step gray value readings plotted against aluminium thickness.‘ The gs of the tooth section images were within the linear portion of the dge curve and linear regreu‘n was used to convert the gray value data into values of equivalent thicknesses of aluminium; The section thicknesses were measured and the % mineral [Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by wilksar ation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar date computed using the equation of Angmer et. at. (1963) and the linear absorption coefficients of aluminium, organic matter pius water and apetitic mineral (131.5, 11.3, and 260.5 respectively). The image of the median strip between the two iesions was scanned six times and ed to give a centre! sound-enamei densitometric profiie.

The iesion images (remineralisetion windows and demineralieation con‘troi windows) to the gingivat and occlusal side of the median strip were simileriy scanned. at; close as possible to the median strip but avoiding any irregularities ly found at the lesion edges, and the % mineral profiles computed.

Example 8 ln this example, a number of ations are provided to exemplify the ways in which complexes of the invention may be tormuieted for different purpose compositions as. described more generally above. These are only exampies of the type of formuiations that may be. provided using various embodiments of the ion.

Toothpaste formulations containing stannous—asmciated stabilized ACP or ACFP Formuiation‘ 1 ingredient % w/v A B C Sorbitol 53,0 53.0 53.0 Silica (Zeodent 119) 20.0 20.0 20.0 Purified water baianoe beianoe e Sodium iauryt sulphate 4.0 4.0 4.0 stenno-us—ese‘ooieted stabilized ACP or 1.2 1.2 2.0 ACFP Sodium monofiuorophoephate 0.3 - — Flavour 1.0 1.0 1.0 Sodium oerboxymethyl cellulose 0.75 0.75 0.75 Titanium dioxide 0.525 0.525 0.525 Xenthen gum 0.47:5 0.475 0.475 Sodinaccherin 0.350 0.350 0.350 [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar pH adjusted to 30 with pho-sphericacid Formulation 2 ingredient % WW “1:: WV ‘24; WW Sorbitol 22.0 22.0 22.0, Irish Moss 1.0 1.0 1.0 Ganirez 19.0 19.0 19.0: Purified water a balance balance Sudium uorophosphate — ~ 0.78 Sadium saccharine 0.3 0.3 0.3 Pyrophosphats 2.0 2.0 2.0 Hydrated alumina 47.0 47.0 47.0.

Flavour 0.05 0.95 0.95 stamens-associated stabilized ACP or 1.0 2.0 2.0 ACFP Sodium lauryl sulphate 2.0 2.0 2.0 pH adjusted to 5—7 with NaOH Furmulation 3 ingredient % wlv Dicalcium phosphate dihyclrate 45.0. al 10.0 Glyceroi 10.0 Sad’ium carboxymethyl cellulose 1.0 Sodium lauryl te '1 .5 Sodiuaauryl sarconiSate 0.5 [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by r [Annotation] wilksar ionNone set by r [Annotation] wilksar Unmarked set by r Flavour 1.0 Sodium saccharine 0.1 Sodium monofluorophosphate 0.3 Chlomexidine gluconate 0.01 Dextranase 0.01 stannous—associated stabilized ACP or ACFP 5.0 Purified water balance pH adjusted in 5-7 with phosphoric acid Formulglion 4 ingredient % wfv tol 22.0 lrish moss 1.0 Gaﬁtriez 19.0 Purified water balance Sodium saccharin 0.3 Pyre'phosphate 2.0 Hydrated alumina 43.0 Sodium manofluerophasphate 0.3 Fiavour 0.95 stannous—associated stabilized NIP or ACFP 5.0 Sodium lauryi sulphate 2.0 pH adjusted to 5.5 with NaOH Formulation 5 lngreb'it % wfv [Annotation] r None set by r [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar Dicalcium phosphate dihydr-ate 45.0 Sorbitol 10.0 Glycerol 10.0 Sodium carboxymsthyl cellulose 1.0 Sodium lauryi sulphate 1.5 Sodium lauryi sarconisate 0.5 Flavour 1.0 Sodium saucharine 0.1 Chlorhexidine gluconate 0.01 Dextranase 0.01 Sodium monofiuorophosphate 0.3 stannous—assuciated stabilized ACP 0r ACFP 5-0 Purified water balance pH adjusted to 5.5 with phosphericacid Formuiation 5 °/o WIV ient Surbitol 53.0 53.0 Silica (Zeddent 119) 20.0 20.0.

Purified water balance balance Sodium l-auryl sulphate 4.0 4.0 stannous—associated stabilized ACP or ACFP 5.0 5.0 Sodium monofluorophosphate 0.3 Sodium dihydrogert phczsphate 1.45 1.45 Flax/0D 1.0 1.0 [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar Sodium oarbdxymethyl cellulose 0.75 0.75 um dioxide (Rutile) 0. 525 0.525 Xanthan gum 0.47.5 0.475 Sodium saccharin 0.350 0.350 Sodium fluoride 0.243 pH adjusted to 5—7 with phosphoric aoidiaOH Formulation 7 ‘34: wiv Ingredient Sor‘bitol 80% solution) 31.0 31.0 Purified water balance e Silica 17.0 17.0 Glycerol 8.0 8.0 Sodium lauryi sulphate 4.0 4.0 Polyethylene glycol 300 1.0 1.0 Sodium fluoride 0.243 Titanium dioxide (Rutile) 0.525 052.5 Xanthan gum 0.4?5 0.47.5 Sodium carboxymethyl cellulose 0.5 0.5 Sodium saccharine 0.286 0.285 Sodium acid pyrophosphat‘e 2.4 2.4 Tetra sodium pyrophosphate 2.2 2.2 r 1.0 1.0 stannoueassmciated stabilized ACP or ACFP 5.0 5.0 SodiLDnonofluorophosphate 0.3 [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar pH adjusted to 5-7 with phosphoric acidiNaOl—t it wiii Ice understood that the invention dieciosed and defined in this specification extends to all alternative combinations of two or more ef the individual features mentioned or t from the text or gs. Aii of these different ations constitute various atternativ‘e aspects of the invention.

REFERENCES . Aoba T, Fejerskov 0(2002). Dentai fiuoro‘sis: try and biology. Crit Rev Oral Biol "Med 13:155-70.

. Den Besten PK, Thariani H (1992). Biologicai mechanisms of fluorosis and ievel and timing of ic re to e with respect to fluorosis. J Dent Res 71:1238~43.

. Fejerskov O, Manji F, Baeium V {1990). The nature and isms of dental fluorosis in man. J Dent Res 69 Spec No:692~700; discussion 721.

. Fejers‘kov 0, Yanagisawe T, Tohda H, Larsen Md, Josephsen K, Moshe HJ (1991). Posteruptive s in human dental fluorosis-«e histotogical and ultrestructurei study. Proc Finn Dent Soc 87:607-153.

. Giambro NJ, ProstakK, Den Besten PK (1995). Characterization of fiuorosed human enamel by color reﬂectance, ultrestructure, and eiementei composition.

Caries Res 29251—7.

- Reynolds EC (1998) Anticariogenic compiexee of amorphous caicinm phosphate stabilized by casein phosphopeptides: a review. Spec Care Dentist 18:8—16.

. Reynolds EC, Cai F, Shen P, Walker GD (2003). Retention in plaque and rem'ineralization of enemei lesions by various forms of ceicium in a m'outhrinse or sugar-free g gum J Dent Res 82206—1 1.

. She-n P; Cai F, Nowi-c‘ki A, Vincent J, Reynoids EC (2001). Reminerelization of enamel subsurface lesions by sugar-tree chewing gum containing casein phosphopeptide-amorphous caicium phosphate. J Dent Res 802065—70. 1003223100

Claims

1. A stannous-associated casein-phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP) and/or -amorphous calcium fluoride phosphate (CPP-ACFP) complex having a stannous ion content of at least 1 mole of stannous per mole of casein-phosphopeptide.

2. A stannous-associated casein-phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP) and/or casein-amorphous calcium fluoride phosphate (CPP-ACFP) complex according to claim 1, wherein the complex has a stannous ion content of at least 5 moles of stannous per mole of casein-phosphopeptide.

3. A stannous-associated casein-phosphopeptide stabilized amorphous m phosphate (CPP-ACP) and/or ous calcium fluoride phosphate (CPP-ACFP) complex according to claim 2, wherein the complex has a stannous ion content of about 7 moles of stannous per mole of caseinphosphopeptide.

4. A stannous-associated casein-phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP) and/or ous calcium fluoride phosphate (CPP-ACFP) complex ing to any one of claim 1 to 3, wherein the stannous is provided by a nd selected from the group consisting of stannous fluoride, us chloride, ium stannous fluoride, sodium stannous fluorozirconate, stannous chloride fluoride, stannous acetate, sodium stannous de, stannous hexafluorozirconate, stannous e, stannous tartrate, stannous gluconate, and disodium monostannous citrate.

5. A stannous-associated casein-phosphopeptide stabilized amorphous calcium phosphate CP) and/or amorphous calcium fluoride phosphate (CPP-ACFP) complex according to claim 4, wherein the compound is stannous fluoride.

6. A stannous-associated casein-phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP) and/or amorphous calcium fluoride phosphate (CPP-ACFP) complex ing to any one of claims 1 to 5, wherein the fluoride is provided by sodium fluoride and stannous fluoride. 1003223100

7. A method of ing a stannous-associated casein-phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP) complex comprising the step of admixing P and a stannous compound in an aqueous solution, while maintaining the pH at about 6.5 or below.

8. A method of producing a stannous-associated casein-phosphopeptide stabilized amorphous calcium fluoride phosphate (CPP-ACFP) complex comprising the step of admixing CPP-ACFP and a us nd in an aqueous solution, while maintaining the pH at about 6.5 or below.

9. A composition including a stannous-associated casein-phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP) and/or amorphous calcium fluoride phosphate (CPP-ACFP) x according to any one of claims 1 to 6.

10. A composition according to claim 9, wherein the composition is in the form applicable to the mouth selected from the group consisting of dentifrice ing toothpaste, toothpowder and liquid dentifrice, mouthwash, mouthrinse, mouth spray, varnish, dental cement, troche, chewing gum, dental paste, gingival massage cream, gargle tablet, dairy product and other foodstuffs.

11. An oral care formulation comprising a stannous-associated phosphopeptide stabilized amorphous m phosphate (CPP-ACP) and/or ous calcium fluoride phosphate (CPP-ACFP) complex according to any one of claims 1 to 6.

12. An oral care formulation according to claim 11, formulated as a dentifrice.

13. Use of a stannous-associated phosphopeptide stabilized CPP-ACP and/or CPP-ACFP complex in the manufacture of a medicament for mineralizing a dental surface or rface.

14. The use according to claim 13, wherein the dental surface is dental enamel or dentine.

15. The use ing claim 13 or 14, n the dental surface is a lesion in the enamel caused by caries, dental erosion or fluorosis. 1003223100

16. The use ing to any one of claims 13 to 15, wherein the dental surface is exposed dentine causing dentinal hypersensitivity.

17. Use of a stannous-associated phosphopeptide stabilized CPP-ACP and/or CPP-ACFP complex in the manufacture of a medicament for forming a protective layer bonded to the surface of the tooth.

18. The use ing to claim 17, wherein the layer has a calcium : phosphate ratio equivalent to normal apatite.

19. The use according to claim 17 or 18, wherein the layer contains peptides from casein phosphopeptides that stabilize the CPP-ACP and/or CPP-ACFP.

20. The use according to any one of claims 17 to 19, wherein the layer contains carbon, oxygen, fluoride, phosphate, calcium and stannous.

21. The use according to claim 20, wherein the layer contains 20 to 30 wt% carbon, 35 to 45 wt% oxygen, 0.1 to 1 wt% fluoride, 8 to 12 wt% phosphate, 16 to 24 wt% calcium and 0.5 to 2 wt% stannous.

22. A method of lizing a dental surface or rface of a non-human animal comprising administering a stannous-associated phosphopeptide stabilized CPP-ACP and/or CPP-ACFP complex to the dental surface or subsurface of the non-human , wherein the non-human animal is a domestic animal such as , sheep, horses and poultry, a companion animal such as cats and dogs, or a zoo animal. [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar