EA043823B1 - COMPLEXES FOR TREATING SENSITIVITY - Google Patents

Description

Cross reference to related applications

The invention claims the benefit of Australian Provisional Patent Application No. 2017900892, the entire contents of which are incorporated herein by reference in their entirety.

Field of invention

The present invention relates to improved complexes of amorphous calcium phosphate and/or amorphous calcium fluoride-phosphate stabilized by phosphopeptides, and compositions containing these complexes. Methods for producing complexes according to the invention and treating or preventing tooth sensitivity are also proposed.

Prior Art

The three mineralized components of teeth are enamel, cementum and dentin. In human teeth, coronal dentin is covered with enamel, while root dentin is covered with cementum. Dentin surrounds the tooth pulp, which provides its blood supply and innervation. Unlike enamel and cementum, dentin is penetrated by a network of tubular structures called dentinal tubules. These tubules are covered by enamel (crown) and cementum (root), which form a layer that protects the pulp from physical and chemical external influences, such as changes in temperature and acid, and prevents them from affecting the nerve processes and increased sensitivity of the dentin. The diameter of the dentinal tubules passing through the dentin layer and opening on its surface varies from 1 to 2.5 µm.

The walls of the tubules in dentin are represented by a calcified dentin matrix, and the internal space of the tubules is filled with fluid (dentinal fluid) originating from the tissue fluid of the pulp and serum. Due to the rigidity of their walls, the fluid filling the narrow dentinal tubules allows cold, tactile, evaporative and osmotic stimuli to be transmitted through the dentin to the pulp in the form of fluid movement. This movement of dentinal fluid is perceived as an acute pain of short duration. This pain occurs when odontoblasts are displaced, penetrating the pulpal ends of the tubules, leading to stimulation of the mechanoreceptors of the pulpal nerve fibers connected to them. This nerve fiber reaction is usually called dentin pain, and the dentin in which it occurs is dentin with hypersensitivity.

Dentin hypersensitivity results from the loss of the enamel or cementum that covers and protects the dentin. Typically, cement breaks down more easily than enamel because it is thinner and less resistant to acids. However, loss of cementum integrity is not possible without prior atrophy of the gingival margin and exposure of the root surface to the oral environment. Individuals with cementum loss and dentin sensitivity often experience pain when the exposed area of the tooth is exposed to cold air, hot or cold liquids, sweet or sour foods, or when touched with a metal object. Patients suffering from dental hypersensitivity have an increased number of open dentinal tubules and/or tubules of larger than normal diameter.

WO 2015/095932 describes complexes having certain beneficial properties. The present invention demonstrates significant advantages over these complexes.

There is a need for compositions and methods for minimizing effects on dentinal tubules to treat and/or prevent dentin sensitivity.

In this invention, reference to any piece of prior art is not an admission or suggestion that that piece of prior art is part of the general knowledge in any jurisdiction or that one of ordinary skill in the art would reasonably be expected to understand this prior art element, will find it important and/or will combine it with other prior art elements.

Summary of the invention

In one aspect, the invention provides a tin-associated phosphopeptide (PP)-stabilized amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complex with a tin ion content equal to or greater than 1 mole of tin per mole of PP, but less than 4 moles of tin per mole of PP.

Preferably, the tin ion content of the complex is from about 1 mol tin per mole PP to about 2 mol tin per mole PP. The tin ion content of the complex may be approximately 1 mol tin per mole PP or approximately 2 mol tin per mole PP. Most preferably, the tin ion content of the complex is approximately 1.6 moles of tin per mole of PP.

In another aspect, the present invention provides a composition comprising a tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized with a phosphopeptide (PP) of the invention.

In another aspect, the present invention provides a composition comprising a complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized with a phosphopeptide (PP) and a tin compound, wherein the content of tin ions in the composition is equal to or greater than 1 mole tin per mole PP, but is less than 4 mol tin per mole PP.

Preferably, the tin ion content of the composition is from about 1 mol tin per mole PP to about 2 mol tin per mole PP. The tin ion content of the composition may be approximately 1 mole of tin per mole of PP or approximately 2 moles of tin per mole of PP. Most preferably, the tin ion content of the composition is approximately 1.6 moles of tin per mole of PP.

Preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect, the present invention also provides a method for treating or preventing dentin sensitivity in an individual in need thereof, comprising administering a tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized with a phosphopeptide (PP) containing ions tin equal to or greater than 1 mol tin per mole PP, but less than 4 mol tin per mole PP, providing treatment or prevention of dentin hypersensitivity in an individual.

In another aspect, the present invention also provides a method for treating or preventing dentin sensitivity in an individual in need thereof, comprising administering a composition comprising a complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized with a phosphopeptide (PP) , and a tin compound, wherein the content of tin ions in the composition is equal to or greater than 1 mol tin per mole PP, but less than 4 mol tin per mole PP, providing treatment or prevention of dentin hypersensitivity in an individual.

Preferably, dentin sensitivity is dentin hypersensitivity.

Preferably, the method further includes the step of identifying an individual in need of treatment. For example, the invention includes, in addition to the steps of any method described herein, the step of identifying a subject suffering from dentin sensitivity, specifically from dentin hypersensitivity.

In another aspect, the present invention provides a method for closing open dentinal tubules in an individual, comprising administering:

a tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized by phosphopeptide (PP), with a tin ion content equal to or greater than 1 mol tin per mole PP, but less than 4 mol tin per mole PP; or a composition containing a complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized with a phosphopeptide (PP) and a tin compound, wherein the content of tin ions in the composition is equal to or greater than 1 mole of tin per mole of PP, but less than 4 moles of tin per mole of PP to provide closure of open dentinal tubules in an individual.

In another aspect, the present invention provides a method of forming a layer over exposed dentinal tubules in an individual, comprising administering:

a tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized by phosphopeptide (PP), with a tin ion content equal to or greater than 1 mol tin per mole PP, but less than 4 mol tin per mole PP; or a composition containing a complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized with a phosphopeptide (PP) and a tin compound, wherein the content of tin ions in the composition is equal to or greater than 1 mole of tin per mole of PP, but less than 4 moles of tin per mole of PP to ensure the formation of a layer over the exposed dentinal tubules in an individual.

Preferably, the method of closing open dentinal tubules or the method of forming a layer over open dentinal tubules further includes the step of identifying open dentinal tubules in an individual.

In another aspect, the present invention provides the use of:

a tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized by phosphopeptide (PP), with a tin ion content equal to or greater than 1 mol tin per mole PP, but less than 4 mol tin per mole PP; or a composition containing a complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized with a phosphopeptide (PP) and a tin compound, wherein the content of tin ions in the composition is equal to or greater than 1 mole of tin per mole of PP, but less than 4 moles of tin per mole of PP, in the preparation of a medicinal product for the treatment or prevention of dentin sensitivity.

Preferably, dentin sensitivity is dentin hypersensitivity.

In another aspect, the present invention provides a tin-associated phosphopeptide (PP) stabilized amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complex with a tin ion content equal to or greater than 1 mole of tin per mole

PP, but less than 4 moles of tin per mole of PP; or a composition containing a complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP), stabilized by a phosphopeptide (PP), and a tin compound, where the content of tin ions in the composition is equal to or greater than 1 mole of tin per mole of PP, but less than 4 moles of tin per mole of PP, for use in the treatment or prevention of dentin sensitivity. Preferably, dentin sensitivity is dentin hypersensitivity.

In any aspect of the invention described herein, the tin ion content is from about 1 mole of tin per mole of PP to about 2 moles of tin per mole of PP. The tin ion content may be approximately 1 mol tin per mole PP or approximately 2 mol tin per mole PP. Most preferably, the tin ion content is approximately 1.6 moles of tin per mole of PP.

In any aspect of the invention, the complex of the invention or the composition of the invention is administered to the oral cavity. Alternatively, it may be administered directly to the area of the mouth in need of treatment, such as an area of exposed dentin. In either case, administration brings the complex or composition into contact with the area of the oral cavity in need of treatment, such as exposed dentin.

In any aspect of the invention, the phosphopeptide is a casein phosphopeptide.

In one embodiment, the tin-associated phosphopeptide (PP)-stabilized amorphous calcium phosphate (ACP) complex consists essentially of or consists of phosphopeptides, tin ions, calcium, phosphate, hydroxide ions and water.

In one embodiment, the tin-associated phosphopeptide (PP)-stabilized amorphous calcium fluoride phosphate (ACFP) complex consists essentially of or consists of phosphopeptides, tin ions, calcium, phosphate, fluoride and hydroxide ions, and water.

In any aspect or embodiment as described herein, the phosphopeptide (PP) stabilized tin-associated ACP or ACFP complex may be formulated with additional calcium phosphate. Typically the composition contains a tin-associated complex of ACP or ACFP stabilized by phosphopeptide (PP), together with at least an equal amount of calcium phosphate by weight.

In any aspect or embodiment of the invention described herein, the composition of the invention or the tin-associated PP-stabilized ACP or ACFP complex of the invention can be applied to the oral cavity, tooth or exposed dentin by the subject in need of treatment or by a dental practitioner.

The tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized with phosphopeptide (PP) of the invention or composition of the invention can be brought into contact with the tooth surface for a period of about 1 to 60, for about 1 -30, approximately 10-60, approximately 10-30, approximately 20-60 or approximately 20-30 min. Preferably, the complex or composition is brought into contact with the tooth surface for approximately 20 minutes.

In another aspect, the present invention provides a method for mineralizing the surface or subsurface of a tooth, comprising contacting the surface or subsurface of a tooth with a tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized by a phosphopeptide (PP) , according to the invention or a composition according to the invention, providing mineralization of the surface or subsurface layer of the tooth.

In a preferred embodiment of the invention, the tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP), stabilized by a phosphopeptide, in the composition has tightly bound and loosely bound calcium, wherein the bound calcium in this complex is less than the tightly bound calcium in the ACP or ACFP complex formed at pH 7.0. It is possible that ACP or ACFP is presented predominantly in the basic form.

In another preferred embodiment, the calcium ion content of the stabilized ACP or ACFP complex or the complex present in the composition exceeds 30 moles of calcium per mole of PP. Preferably, the calcium ion content of the stabilized ACP or ACFP complex is in the range of about 30 to 100 moles of calcium per mole of PP. More preferably, the calcium ion content ranges from about 30 to about 50 moles of calcium per mole of PP.

The invention also relates to a kit for the treatment or prevention of dentin hypersensitivity, containing:

(a) a composition according to the invention, or (b) a tin-associated phosphopeptide-stabilized ACP or ACFP complex according to the invention.

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Preferably, the phosphopeptide stabilized tin-associated ACP or ACFP complex is presented in a pharmaceutically acceptable carrier. Desirably, the kit further contains instructions for their use to treat or prevent dentin hypersensitivity in an individual in need of such treatment. In one embodiment, the composition and complex are present in suitable amounts for treating an individual.

The composition or kit of the invention may further comprise a source of fluoride ions. Fluoride ions can be from any suitable source. The source of fluoride ions may include free fluoride ions or fluoride salts. Examples of fluoride ion sources include, but are not limited to, the following: sodium fluoride, sodium monofluorophosphate, stannous fluoride, sodium fluorosilicate and amino fluoride. They may be present in solution (usually an aqueous solution) or in suspension.

As used herein, unless the context otherwise requires, the term contain and variations of the term such as containing, contains and contained are not intended to exclude other additives, components, elements or steps.

Other aspects of the present invention and other 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 accompanying drawings.

Brief description of graphic materials

Fig. 1. Scanning electron microscopy (SEM) of patient's dentinal tubules treated with distilled deionized water (DDW). Magnification 15000x, scale bar shown at 10 µm. Open dentinal tubules are clearly visible.

Fig. 2. SEM of a patient's dentinal tubules treated with Sn/CPP at a molar ratio of 1.6:1, showing how self-assembled nanofilaments on the dentin surface seal the tubules. Magnification 15000x, scale bar shown at 10 µm.

Fig. 3. SEM of a patient's dentinal tubules treated with Sn/CPP at a molar ratio of 1.6:1, showing how self-assembled nanofilaments on the dentin surface seal the tubules. A separate dentinal tubule is shown. Magnification 60000x, scale bar shown at 2.0 µm.

Fig. 4. SEM of a patient's dentinal tubules treated with Sn/CPP at a molar ratio of 1.6:1, showing how self-assembled nanofilaments on the dentin surface seal the tubule. A separate dentinal tubule (different from that shown in Fig. 3) is shown. Magnification 60000x, scale bar shown at 2.0 µm.

Fig. 5. SEM of patient's dentinal tubules treated with Sn/CPP at a molar ratio of 4:1, showing partial formation of self-assembled nanofilaments on the dentin surface and partial closure of the tubules. Magnification 15000x, scale bar shown at 10 µm.

Fig. 6. SEM of a patient's dentinal tubules treated with Sn/CPP at a molar ratio of 8.6:1, showing partial self-assembly of nanofilaments on the dentin surface and partial closure of any tubules. Magnification 15000x, scale bar shown at 10 µm.

Detailed description of embodiments

It should be understood that the invention as disclosed and defined herein covers all alternative combinations of two or more individual features indicated or apparent from the text or graphics presented. All of these different combinations constitute various alternative aspects of the invention.

Other aspects of the present invention and other 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 accompanying drawings.

Certain embodiments of the invention will now be described in detail. Although the invention will be described in connection with specific embodiments, it should be understood that these embodiments are not given to limit the invention. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents that 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 that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

All patents and publications mentioned herein are incorporated by reference into this invention in their entirety.

For the purpose of correct interpretation of this invention, terms used in the singular also include the plural, and vice versa.

As used herein, unless the context otherwise requires, the term contain and variations of the term such as containing, contains and contained are not intended to exclude other additives, components, elements or steps. As used herein, unless the context otherwise requires, the terms contain and include may be used interchangeably.

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The present invention is based on the unexpected discovery that a tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized with phosphopeptide (PP), with a tin ion content equal to or greater than 1 mole of tin per mole of PP, but less than 4 mol tin per mole PP, can form self-assembled nanofilaments that can seal dentinal tubules. Specifically, self-assembled nanofilaments are formed to such an extent that they completely cover the openings of the dentinal tubules, forming a network of nanofilaments on the dentin surface. This does not happen when the content of tin ions is more than 4 mol per mole of phosphopeptide.

Without regard to any theory or mechanism of action, it is proposed that tin ions and phosphopeptides interact with the dentin surface, resulting in a unique surface coverage pattern of a self-assembled network of nanofilaments on the dentin surface covering the tubules. These nanofilaments are believed to be a phosphopeptide cross-linked with tin, releasing the calcium, phosphate and fluoride ions it contains to the surface to form the nanofilaments. The results reported here indicate that a molar ratio equal to or greater than 1 mol but less than 4 mol tin per mole of phosphopeptide is critical for the formation of cross-linked nanofilaments and thus greater reduction of dentin hypersensitivity.

The tin compound or tin compound may be any soluble tin compound suitable for oral administration. Preferably, the tin-containing compound is a tin salt. Tin salt may contain fluoride. Stannous salt includes, but is not limited to, stannous fluoride, stannous chloride, potassium stannous fluoride, sodium stannous fluorozirconate, stannous chloride fluoride, stannous acetate, sodium stannous fluoride, stannous hexafluorozirconate, stannous sulfate, stannous tartrate, stannous gluconate, disodium citrate and tin. Preferred tin salts include stannous fluoride and stannous chloride.

Tin may be associated with amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) stabilized phosphopeptide as determined using the experimental protocol in Example 2. In one embodiment, the tin-associated PP stabilized amorphous calcium phosphate (ACP) complex and/ or amorphous calcium fluoride phosphate (ACFP) is prepared by the method as described herein, including, without limitation, the method described in Example 1.

In any aspect of the invention, the tin ion content of the complex of the invention may be equal to or greater than 1 mol tin per mole PP, but less than 4 mol tin per mole PP. For example, the tin ion content may be equal to or greater than approximately 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 , 3.0, 3.2, 3.4, 3.6, 3.8 or 4.0 mol tin per mole PP. Also implied is a range of tin ion contents specified by any 2 values of 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2, 8, 3.0, 3.2, 3.4, 3.6, 3.8 or 4.0.

In any aspect of the invention, the tin ion content of the complex of the invention may range from about 1 mole of tin per mole of PP to about 3 moles of tin per mole of PP.

In any aspect, the tin ion content of the complex is from about 1 mole of tin per mole of PP to about 2 moles of tin per mole of PP. This range includes 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mol tin per mole PP. The tin ion content of the complex may be approximately 1 mol tin per mole PP or approximately 2 mol tin per mole PP.

In any aspect of the invention, the tin ion content of the complex of the invention may range from about 1.2 mol tin per mole PP to about 1.8 mol tin per mole PP. Preferably, the tin ion content is from about 1.4 mol tin per mole PP to about 1.8 mol tin per mole PP; or from about 1.3 mol tin per mole PP to about 1.5 mol tin per mole PP. Most preferably, the tin ion content of the complex is approximately 1.6 moles of tin per mole of PP.

In addition, the content of tin ions in the composition of the invention may be equivalent to the complex of the invention. For example, the tin ion content of the composition of the invention may be 1.6 moles of tin per mole of PP.

Accordingly, when used herein, reference to the tin ion content of the complex may also refer to the tin ion content of the composition.

The tin ion content of the complex or composition can be determined by any method as described herein or known in the art.

In any aspect, the present invention is applied to a tooth surface or area of the oral cavity, which may be tooth enamel or dentin. Typically the surface is exposed dentin, which may be exposed by destruction of the enamel or cementum. Exposed dentin preferentially results in dentin sensitivity. Exposed dentin resulting from cementum failure can be identified by atrophy of the gingival margin and exposure of the root surface to the oral environment. Typically the oral cavity site is located on the molar.

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Typically, open dentin contains dentinal tubules, the diameter of which exceeds

1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4 or 2.5 µm.

An individual suffering from dentin sensitivity, preferably hypersensitivity, may be an individual experiencing pain when thermal or tactile stimuli are applied to an area of a tooth. Specifically, an individual may experience pain when the tooth area comes into contact with cold air, hot and cold liquids, sweet or sour foods, or when touched with a metal object. In any aspect, a subject may be identified for treatment by exposure to any of these stimuli prior to treatment to determine whether the subject is experiencing pain sensations. An individual with dental hypersensitivity may be identified as an individual who has an increased number of open dentinal tubules and/or larger than normal diameter tubules, for example, an individual may have dentinal tubules whose opening diameters exceed 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4 or 2.5 µm, or have a greater number of such tubules.

The words treat or treatment refer to therapeutic treatment whose goal is to slow down (reduce) an unwanted physiological change or disorder. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in severity of the condition, stabilized (i.e., not worsening) state of the condition, delay or slowing of progression of the condition, alleviation of the disease/condition, and remission (partial or complete), detectable or undetectable. Treatment does not necessarily result in a complete absence of detectable symptoms of the condition, but may reduce or minimize the complications and side effects of the condition. Monitoring the effectiveness or ineffectiveness of treatment can be done using a physical examination of the individual or by response to any thermal, tactile or chemical stimulation, as described here. Preferably, the individual experiences a decrease in pain intensity or temporal frequency of pain. Methods for identifying individuals with varying degrees of dentin sensitivity and assessing the effectiveness of treatment or prevention are described here and also include the methods described in Med Oral Patol Oral Cir Bucal. 2008 Mar 1;13(3):E201-6. Treatment of an individual can be assessed by comparing the level of pain experienced upon exposure to any of the stimuli described herein before and after treatment, where a decrease in pain after treatment indicates a decrease in sensation.

The words warn and caution usually refer to prophylactic or preventative measures taken to protect or prevent an individual who is not sensitive from developing sensitivity. Individuals who may require prophylaxis are individuals undergoing a dental procedure, particularly a dental procedure that results in exposed dentin.

In any aspect of the present invention, the tin ion content indicated above may be the content of tin ions tightly bound to the complex (as described herein). When assessing the tin ion content, the content of tightly bound tin ions is measured by the methods described herein, particularly in Example 2.

The invention provides a tin-associated complex of ACP and/or ACFP, stabilized with a phosphopeptide, containing tin ions, which remain associated with the complex after centrifugation through a filter with a molecular weight cutoff of 1000 at approximately 3000 g for 1 hour at room temperature, where the ion content tin that remains associated with the complex is equal to or greater than 1 mol tin per mole PP, but less than 4 mol tin per mole PP.

The invention also provides a tin-associated ACP or ACFP complex stabilized by a phosphopeptide, wherein at least 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99 or 100% tin is tightly bound to the complex, as determined by the method described in Example 2.

In any composition of the invention, the amount of ACP or ACFP stabilized by a phosphopeptide, preferably CPP-ACP or ACFP, may be from about 2 to 5%, and the amount of tin compound, preferably SnF ₂ , may be 500 ppm F The amount of ACP or ACFP stabilized with phosphopeptide may be 2 or 5%.

In any composition of the invention, the amount of ACP or ACFP stabilized by a phosphopeptide, preferably CPP-ACP or ACFP, may be equal to or greater than about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3. 5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10% by weight. Typically the amount of tin compound, preferably SnF ₂ , may be equal to or greater than about 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440 , 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 2000, 3000, 4000 or 5000 ppm. F.

In one aspect, the present invention provides a method for mineralizing the surface or subsurface of a tooth, comprising contacting the surface or subsurface of a tooth with a tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) stabilized by a phosphopeptide (PP) , according to the invention or a composition according to the invention, providing mineralization of the surface or subsurface layer of the tooth.

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In a preferred embodiment, the tin-associated phosphopeptide-stabilized amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complex has tightly bound and loosely bound calcium, wherein the bound calcium in this complex is less than the tightly bound calcium in the ACP complex or ACFP formed at pH 7.0. Maybe,

ACP or ACFP is presented primarily in its basic form.

In another aspect, the invention also provides a composition comprising tin-associated ACP or ACFP stabilized with a phosphopeptide, further comprising fluoride, wherein the fluoride is in the form of stannous fluoride and/or sodium fluoride. Preferably, for every mole of tin there are 2 moles of fluoride. Preferably, the composition is a toothpaste or any other oral care composition as described herein.

In a preferred embodiment, the calcium ion content of the tin-associated phosphopeptide stabilized ACP or ACFP complex is in the range of about 30 to 100 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.

In a preferred embodiment, the tin-associated phosphopeptide-stabilized ACP and/or ACFP complex is in the form of a casein phosphopeptide-stabilized ACP and/or ACFP complex.

In any aspect of the invention, the phosphopeptide or phosphoprotein is a casein phosphopeptide or phosphoprotein or a tryptic cleavage product thereof.

Preferably, the ACP phase is predominantly (ie, more than 50%) a basic phase, wherein the ACP contains primarily Ca ²⁺ , PO4 ^{3 -} and OH ^- ions. The main phase of ACP may have the general formula [Ca3(PO ₄ ) ₂ ] _x [Ca ₂ (PO ₄ )(OH)], where x is 1 or more. Preferably, x is from 1 to 5. More preferably, x is 1, that is, the two components of said formula are present in equal proportions. Accordingly, in one embodiment, the main phase of ACP has the formula Ca3(PO4)2Ca2(PO4)(OH).

Preferably, the ACFP phase is predominantly (ie, more than 50%) a basic phase, where the ACFP contains primarily Ca ²⁺ , PO4 ^{3 -} and F ^- ions. The main phase of ACFP may have the general formula [Ca3( _PO4 ) ₂ ] _x [ _Ca2 ( _PO4 )F] _y , where x is 1 or more when y is 1, or where y is 1 or more when x is 1. Preferably, y is 1 and x is from 1 to 3. More preferably, y is 1 and x is 1, that is, the two components of said formula are present in equal proportions. Accordingly, in one embodiment, the main phase of ACFP has the formula Ca3(PO4)2Ca2(PO4)F.

When referred to herein, the tin-associated PP-stabilized ACP or ACFP complex includes the tin-associated PP-stabilized ACP or ACFP complex formed at a pH of 7.3 or less. Preferably, the complex is formed at a pH ranging from about 5.0 to slightly less than 7.3. More preferably, the complex is formed at a pH ranging from about 4.0 to 7.3 or from 5.0 to about 6.0. In one embodiment, the pH is maintained at 7.3 or less during complex formation. In a preferred embodiment, the complex is formed at a pH of approximately 5.5. Preferably, the ACP or ACFP complex is present predominantly in its base form. When preparing a tin-associated stabilized complex of ACP or ACFP on an industrial scale, it may be prepared as a bulk solution, the pH of which may be higher than about 7.3, preferably about 9.0, however, the local pH during complex formation is less than than about 7.3, preferably from about 4.0 to 6.5, preferably about 5.5.

When producing tin-associated PP-stabilized ACP or ACFP or stabilized ACP or ACFP in the laboratory, in smaller quantities than when it is produced industrially, it is possible to maintain the desired pH value of the entire solution, that is, if CPP-ACP was obtained at pH 5, 5, the pH of the entire solution was maintained at 5.5 during the formation of CPP-ACP. However, in commercial production, reducing the pH of the entire bulk solution to 5.5 may be neither necessary nor desirable since the only part of the bulk solution where an acidic pH is needed is the complexation region, and the bulk solution can and does have local pH fluctuations . In particular, pH fluctuations arise from the protons provided by a phosphate compound, such as dihydrogen phosphate, since its addition releases protons from acidic phosphate ions as they are converted to the basic form PO ₄ ^3- . Thus, although the overall pH of the bulk solution may be greater than 7.3, such as approximately 9, the local pH at which CPP-ACP formation occurs is lower and is typically less than 7.3 or 6.5, preferably approximately from 4.0 to 7.3, more preferably about 5.5. These fluctuations are local in nature due to the volume of the bulk solution.

The present invention also provides a method for producing the tin-associated PP-stabilized ACP of the invention, comprising the steps of:

(i) obtaining a solution containing at least one phosphopeptide; and (ii) mixing solutions containing calcium ions, phosphate ions and hydroxide ions, with

- 7 043823 maintaining a pH of approximately 7.3 or less; and (iii) admixing a tin compound; or (i) providing a solution of ACP stabilized with a phosphopeptide; and (ii) admixing a tin compound.

The present invention also provides a method for producing tin-associated PP-stabilized ACFP, comprising the steps of:

(i) obtaining a solution containing at least one phosphopeptide; and (ii) admixing solutions containing calcium ions, phosphate ions, hydroxide ions and fluoride ions while maintaining a pH of approximately 7.3 or less; and (iii) admixing a tin compound; or (i) providing a solution of ACFP stabilized with a phosphopeptide; and (ii) admixing a tin compound.

To maintain a virtually constant pH of a phosphopeptide solution, hydroxide ions can be titrated into it. Calcium and phosphate ions can be titrated into the phosphopeptide solution with constant stirring and at a rate that avoids the formation of calcium phosphate precipitate in the phosphopeptide solution.

The present invention also provides a method for producing the tin-associated PP-stabilized ACP of the invention, comprising the steps of:

(i) obtaining a solution containing at least one phosphopeptide; and (ii) mixing solutions containing calcium ions and phosphate ions while maintaining a pH of approximately 7.3 or less; and (iii) admixing a tin compound; or (iv) providing a solution of ACP stabilized with a phosphopeptide; and (v) admixing a tin compound.

Preferably, the method does not involve adding any base or acid. For example, hydroxide ions are not added separately to a solution containing calcium ions, phosphate ions or a tin compound.

The present invention also provides a method for producing the tin-associated PP-stabilized ACFP of the invention, comprising the steps of:

(i) obtaining a solution containing at least one phosphopeptide; and (ii) mixing solutions containing calcium ions, phosphate ions and fluoride ions while maintaining a pH of about 7.3 or less; and (vi) admixing a tin compound; or (iii) providing a solution of ACFP stabilized with a phosphopeptide; and mixing in a tin compound.

Preferably, the method does not involve adding any base or acid. For example, hydroxide ions are not added separately to a solution containing calcium ions, phosphate ions, fluoride ions or a tin compound.

The tin-associated PP-stabilized ACP of the invention can be prepared by a process including the step of admixing CPP-ACP and a tin compound in an aqueous solution while maintaining a pH of about 7.3 or less.

The tin-associated PP-stabilized ACFP of the invention can be prepared by a process including the step of admixing CPP-ACFP and a tin compound in an aqueous solution while maintaining a pH of about 7.3 or less.

The tin-associated PP-stabilized ACP of the invention can be prepared by a process comprising the steps of:

(i) obtaining a solution containing CPP-ACP; and (ii) admixing the tin compound while maintaining a pH of about 7.3 or less.

The tin-associated stabilized ACFP of the invention can be prepared by a process comprising the steps of:

(i) obtaining a solution containing CPP-ACFP; and (ii) admixing the tin compound while maintaining a pH of about 7.3 or less.

Preferably, the tin compound is tin fluoride. Optionally, methods for producing tin-associated PP-stabilized ACP or tin-associated PP-stabilized ACFP further include admixing sodium fluoride in step (ii).

If necessary, the pH can be maintained using an acid such as HCl.

Preferably, a solution containing CPP-ACP or CPP-ACFP is prepared by adding CPP-ACP or CPP-ACFP to distilled or deionized water. When using a solution containing 2% or more, preferably 5%, CPP-ACP or CPP-ACFP, there is no need to add any base or acid during the process.

The tin-associated PP-stabilized complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) according to the invention can be prepared by mixing PP-8 043823 stabilized complex of amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) with stannous fluoride.

In any method described herein for producing tin-associated PP-stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) complex, the amount of ACP or ACFP stabilized by a phosphopeptide, preferably CPP-ACP or ACFP, may be greater than or equal to 2 or 5%, and the amount of tin compound, preferably SnF ₂ , may be greater than or equal to 500 ppm. F.

The tin-associated PP-stabilized ACP or ACFP complex as described herein may take the form of the closed complex shown in FIG. 2 in Cross et al., 2007. Current Pharmaceutical Design, 13, 793-800.

The composition comprising the tin-associated PP-stabilized ACP and/or ACFP complex may further contain at least an equal amount by weight of calcium phosphate. Preferably, the calcium phosphate is CaHPO ₄ . Preferably, calcium phosphate (eg CaHPO ₄ ) is dry mixed with the PP-stabilized ACP and/or ACFP complex. In a preferred embodiment, the ratio of PP-ACP and/or PP-ACFP complex: calcium phosphate is about 1:1-50, more preferably about 1:1-25, more preferably about 1:5-15. In one embodiment, the ratio of PP-ACP and/or PP-ACFP complex: calcium phosphate is approximately 1:10.

Preferably, the dry mix form of calcium phosphate is any soluble calcium phosphate, including, but not limited to, CaHPO ₄ , CaHPO ₄ and calcium lactate.

The composition as described herein may additionally contain free fluoride ions. Fluoride ions can be from any suitable source. The source of fluoride ions may include free fluoride ions or fluoride salts. Examples of fluoride ion sources include, but are not limited to, the following: sodium fluoride, sodium monofluorophosphate, stannous fluoride, sodium fluorosilicate and amino fluoride. They may be present in solution (usually an aqueous solution) or in suspension.

Fluoride ions are preferably present in the composition in an amount greater than 1 ppm. More preferably, the amount is greater than 3 ppm. In another embodiment it is preferably greater than 10 ppm. In typical embodiments described below, this amount may be from 100 to 5000, preferably from 400 to 3000, preferably from 1000 to 300 ppm. The fluoride content of oral compositions is most often measured in ppm. methods commonly used in the art. Where fluoride is from a stabilized ACP source, ppm. refers to the concentration of fluoride in that source, usually in a solution or suspension of biologically available fluoride.

As used herein, a phosphopeptide refers to an amino acid sequence in which at least one amino acid is phosphorylated. Preferably, the phosphopeptide contains one or more amino acid sequences -A-B-C-, wherein A is a phosphoamino acid residue, B is any amino acid residue, including a phosphoamino acid residue, and C is selected from glutamyl, aspartyl or a phosphoamino acid residue. Any of the phosphoamino acid residues may independently be a phosphoserine residue. B is desirably a residue whose side chain is neither relatively large nor hydrophobic. It may be Gly, Ala, Val, Met, Leu, Ile, Ser, Thr, Cys, Asp, Glu, Asn, Gln or Lys.

In another embodiment, at least two of the phosphoamino acids present in the sequence are preferably located next to each other. Preferably, the phosphopeptide contains the sequence A-B-C-D-E, where A, B, C, D and E are independently phosphoserine, phosphothreonine, phosphotyrosine, phosphohistidine, glutamic acid or aspartic acid, and at least two, preferably three, of A, B, C, D and E are phosphoamino acids. In a preferred embodiment, the phosphoamino acid residues are phosphoserine residues, most preferably three phosphoserine residues located one behind the other. It is also preferred that D and E are independently glutamic or aspartic acid.

In one embodiment, ACP or ACFP is stabilized by casein phosphopeptide (CPP) present in the form of intact casein or a casein fragment, and the resulting complex preferably has the formula [CPP(ACP)8] _n or [(CPP)(ACFP)8] _n , where n equal to 1 or more, for example 6. The complex formed may be a colloidal complex where aggregation of the main particles occurs to form large (eg 100 nm) colloidal particles suspended in water. Thus, PP may be a casein protein or a phosphopeptide.

PP can come from any source; it may be present in the context of a larger polypeptide, including a full-length casein polypeptide, or it may be isolated by tryptic or other enzymatic or chemical digestion of casein or other high phosphoamino acid proteins such as phosphitin, or by chemical or recombinant synthesis, provided that it contains the sequence -A-B-C- or A-B-C-D-E, as described above.

- 9 043823

The sequence flanking this central sequence can be anything. However, the flanking sequences a _s1 (59-79), β (1-25), a _s2 (46-70) and a _s2 (1-21) are preferred.

The flanking sequences may optionally be modified by deletion, addition or conservative substitution 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 the table below._____________________

Original balance Examples conservative substitutions Preferred conservative substitutions Ala Val, Leu, lie Val Asn Gln Lys His Phe Gln Gln Asn Asn Gly Pro Pro lie Leu, Val, Met, Ala, Phe Leu Leu lie, Val, Met, Ala, Phe lie Lys Arg, Gln, Asn Arg Phe Leu, Val, lie, Ala Leu Pro Gly Gly Ser Thr Thr Val lie, Leu, Met, Phe, Ala Leu Asp Glu Glu Thr Ser Ser Trp Tyr Tyr Tyr Trp Phe Thr Ser Phe

The flanking sequences may also contain amino acid residues not found in nature. Commonly used amino acids that are not genetically coded include:

2-aminoadipic acid (Aad) instead of Glu and Asp;

2-aminopimelic acid (Apm) instead of Glu and Asp;

2-aminobutyric (Abu) acid instead of Met, Leu and other aliphatic amino acids;

2-aminoheptanoic acid (Ahe) instead of Met, Leu and other aliphatic amino acids;

2-aminoisobutyric acid (Aib) instead of Gly;

cyclohexylalanine (Cha) instead of Val, Leu and Ile;

homoarginine (Har) instead of Arg and Lys;

2,3-diaminopropionic acid (Dpr) instead of Lys, Arg and His;

N-ethylglycine (EtGly) instead of Gly, Pro and Ala;

N-ethylasparagine (EtAsn) instead of Asn and Gln;

hydroxylysine (Hyl) instead of Lys;

allohydroxylysine (AHyl) instead of Lys;

3- (and 4-) hydroxyproline (3Hyp, 4Hyp) instead of Pro, Ser and Thr;

alloisoleucine (Alle) instead of Ile, Leu and Val;

ρ-amidinophenylalanine instead of Ala;

N-methylglycine (MeGly, sarcosine) instead of Gly, Pro, Ala;

N-methylisoleucine (MeIle) instead of Ile;

norvaline (Nva) instead of Met and other aliphatic amino acids;

norleucine (Nle) instead of Met and other aliphatic amino acids;

ornithine (Orn) instead of Lys, Arg and His;

citrulline (Cit) and methionine sulfoxide (MSO) instead of Thr, Asn and Gln;

N-methylphenylalanine (MePhe), trimethylphenylalanine, halogen-(F, Cl, Br and I) phenylalanine, trifluorophenylalanine instead of Phe.

In one embodiment, PP is one or more phosphopeptides selected from the group consisting of a _s1 (59-79) [1], β (1-25) [2], a _s2 (46-70) [3] and a _s2 (1-21) [4]:

- 10 043823

[1] Gln ⁵⁹ -Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ile-Val-Pro-AsnSer(P) -Val-Glu-Gln-Lys ⁷⁹ a _s i(59-79);

[2] Arg ¹ -Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)Ser(P)-Ser( P)-Glu-Glu-Ser-Ile-Thr-Arg ²⁵ β(1-25);

[3] Asn ⁴⁶ -Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-Ser(P)-Ser(P)-Ser(P)-Glu-GluSer(P)-Ala-Glu- Val-Ala-Thr-Glu-Glu-Val-Lys ⁷⁰ a _S 2(46-70);

[4] Lys ¹ -Asn-Thr-Met-Glu-His-Val-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-He-Ile-Ser(P)Gln-Glu -Thr-Tyr-Lys ²¹ a _s2 (l-21).

In another embodiment of the invention, the tin-associated PP-stabilized ACP complex and/or the tin-associated PP-stabilized ACFP complex are included in oral compositions such as toothpaste, mouthwashes, or oral compositions to promote prevention and/or treatment of dentin hypersensitivity. Compositions for oral administration contain an amount of tin-associated PP stabilized ACP and/or ACFP sufficient to close open dentinal tubules or to form a layer over open dentinal tubules. Tin-associated PP stabilized ACP and/or ACFP complexes may constitute from 0.01 to 50% of the composition by weight, preferably from 1.0 to 50, preferably from 1.0 to 30, preferably from 1.0 to 20, preferably from 1 0 to 10, preferably 2 to 10% of the composition by weight. In a particularly preferred embodiment, the oral composition of the present invention contains approximately 2% stabilized ACP or ACFP complexes or mixtures thereof.

The oral composition of the present invention containing the complex of the invention can be prepared and used in various forms applicable to the oral cavity, such as dentifrices, including toothpastes, toothpowders and liquid dentifrices, rinses oral rinses, mouth sprays, dental varnish, dental cement, lozenges, chewing gums, dental pastes, gum massage creams, mouthwash tablets, dairy products and other foods including yogurt. The oral composition of this invention may further contain well-known additional ingredients, depending on the type and form of the particular oral composition. Certain compositions of the invention, such as toothpastes, toothpowders and liquid dentifrices, mouthwashes, oral rinses and oral sprays, have relatively low viscosity.

A dentifrice or paste for topical application to a sensitive area of a tooth, such as a decayed cementum root surface exposed to the oral cavity, may be a soft applicator or paste. Such dentifrice or paste may or may not contain conventional polishing agent, foaming agent and flavoring agents.

In certain preferred forms of the invention, the oral composition may be substantially liquid, such as a mouthwash, rinse, or spray. In such a preparation, the excipient is usually a water-alcohol mixture, preferably containing a humectant, as described below. Typically the ratio of water to alcohol by weight ranges from about 1:1 to about 20:1. The total amount of water-alcohol mixture in this type of preparation typically ranges from about 70 to about 99.9% by weight of the preparation. The alcohol is usually ethanol or isopropanol. Ethanol is preferred.

In other preferred forms of the present invention, the oral composition may be substantially solid or paste-like, such as a toothpowder, dental tablet, or toothpaste (dental cream), or gel dentifrice. The filler of such solid or paste oral preparations typically contains a dentally acceptable polishing agent. Examples of polishing agents include water-insoluble sodium metaphosphate, potassium metaphosphate, tricalcium phosphate, calcium phosphate dihydrate, anhydrous calcium phosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesium phosphate, calcium carbonate, alumina hydrate, calcined alumina, aluminum silicate, zirconium silicate, silica, bentonite and mixtures thereof. Other suitable polishing agents include thermosetting resin particles such as melamine, phenol and urea formaldehyde resins, and cross-linked polyepoxides and polyesters. Preferred polishing agents include crystalline silica with particle sizes up to about 5 μm, average particle size up to about 1.1 μm, and surface area up to about 50,000 cm /g, silica gel or colloidal silica, and amorphous alkali metal aluminosilicate complexes.

When using clear gels, colloidal silica polishes such as those commercially available under the trade names SYLOID Syloid 72 and Syloid 74 are particularly useful.

- 11 043823 or commercially available under the trade name SANTOCEL Santocel 100, and alkali metal aluminosilicate complexes because their refractive index is close to that of gelling agent-liquid systems (containing water and/or humectant) commonly used in dentifrices .

Many of the so-called water-insoluble polishes are anionic in nature and also contain small amounts of soluble substances. Thus, insoluble sodium metaphosphate may be prepared by any suitable method, for example as shown in Thorpe's Dictionary of Applied Chemistry, Volume 9, 4th Edition, pp. 510-511. Other examples of suitable substances are the insoluble sodium metaphosphate forms known as Madrell's salt and Carroll's salt. These metaphosphate salts exhibit only minor solubility in water and are therefore commonly referred to as insoluble metaphosphates (IMPs). They are present in small quantities in soluble phosphates as impurities, usually in amounts of a few percent, for example up to 4% by weight. If desired, the amount of soluble phosphate, which in the case of insoluble metaphosphate is expected to include soluble sodium trimetaphosphate, can be reduced or eliminated by washing in water. Insoluble alkali metal metaphosphates are usually used in powder form with a particle size such that no more than 1% of the substance has a size greater than 37 microns.

Polishing agents are typically present in solid or paste compositions in concentrations of from about 10 to about 99% by weight. Preferably, they are present in the toothpaste in amounts of from about 10 to about 75%, and in the tooth powder in amounts from about 70 to about 99%. In toothpastes, when using silicon polishing agents, they are usually present in amounts of approximately 10-30% by weight. Other polishing agents are typically present in amounts of approximately 30-75% by weight.

In a toothpaste, the liquid filler may typically contain water and a humectant in an amount of from about 10 to about 80% by weight of the formulation. Examples of suitable humectants/carriers are glycerin, propylene glycol, sorbitol and polypropylene glycol. Liquid mixtures of water, glycerin and sorbitol are also useful. In transparent gels where refractive index is important, approximately 2.5-30% w/w is preferably used. water, from 0 to about 70% wt./mass. glycerol and approximately 20-80% wt./wt. sorbitol

Toothpaste, creams and gels typically contain a natural or synthetic thickener or gelling agent in a proportion of from about 0.1 to about 10, preferably from about 0.5 to about 5% w/w. A suitable thickening agent is synthetic hectorite, a synthetic clay with a colloidal complex of magnesium and alkali metal silicates, available, for example, in the form of laponite (eg CP, SP 2002, D), commercially available from Laporte Industries Limited. Laponite D is approximately 58.00%, by weight, SiO ₂ , 25.40% MgO, 3.05% Na ₂ O, 0.98% Li ₂ O with some water and trace metals. Its true density is 2.53 and its apparent bulk density is 1.0 g/ml at 8% moisture content.

Other suitable thickeners include Irish moss, iota-carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutylmethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose (e.g., available in the form of natrozole), sodium carboxymethylcellulose, and colloidal silica such as micronized siloid (e.g., 244) . Also included may be solubilizing agents such as wetting polyols such as propylene glycol, dipropylene glycol and hexylene glycol, cellosolves such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing at least about 12 straight chain carbon atoms such as olive oil, castor oil and petrolatum, and esters such as amyl acetate, ethyl acetate and benzyl benzoate.

It should be understood that, as a matter of general practice, oral preparations will generally be sold or otherwise distributed in suitable labeled packaging. Thus, a container of mouthwash will have a label describing it essentially as a mouthwash or rinse and instructions for its use; and the toothpaste, cream or gel will usually be in a flexible tube, typically made of aluminum, thin lead or plastic, or other external pressure dispenser, pump or pressure dispenser for dispensing the contents, labeled with a description thereof, according to essentially like toothpaste, gel or dental cream.

Organic surfactants can be used in the compositions of the present invention to achieve greater preventive effect, help achieve complete distribution of the active agent throughout the oral cavity and increase the cosmetic acceptability of these compositions. The organic surfactant is preferably anionic, non-ionic or ampholytic in nature and preferably does not interact with the active agent. It is preferable to use a detergent as a surfactant that provides foaming of the composition and its cleansing effect. Suitable examples of anionic surfactants are water-soluble salts of monosulfated higher fatty acid monoglycerides such as sodium salt of hydrogenated coconut oil monosulfated fatty acid monoglyceride, higher alkyl sulfates such as sodium lauryl sulfate, alkylaryl sulfonates such as sodium dodecylbenzenesulfonate, higher alkyl sulfoacetates , esters of higher fatty acids and 1,2-dihydroxypropanesulfonate and preferably saturated higher aliphatic acylamides of lower aliphatic aminocarboxylic acids, for example, where the fatty acid has from 12 to 16 carbon atoms, alkyl or acyl radicals and the like. Examples of the latter mentioned amides are N-lauroyl sarcosine and the sodium, potassium and ethanolamine salts of N-lauroyl-, N-myristoyl- or N-palmitoyl sarcosine, which should be substantially free of soap or similar fatty acid compounds. The use of these sarcosine compounds in the oral compositions of the present invention is particularly beneficial because these substances have a long-lasting and potent effect of inhibiting the formation of acids in the oral cavity due to the breakdown of carbohydrates, as well as to some extent reducing the solubility of tooth enamel in acidic solutions. Examples of suitable water-soluble nonionic surfactants include condensation products of ethylene oxide with various reactive hydrogen-containing compounds that are reactive with ethylene oxide and have long hydrophobic chains (e.g., aliphatic chains of about 12 to 20 carbon atoms), the condensation products of which (ethoxamers) contain hydrophilic polyoxyethylene groups, such as condensation products of polyethylene oxide with fatty acids, fatty alcohols, fatty amides, polyhydric alcohols (for example, sorbitan monostearate) and polypropylene oxide (for example, pluronics).

The surfactant is typically present in an amount of about 0.1-5% by weight. It should be noted that the surfactant can help dissolve the active agent of the invention and thereby reduce the required amount of solubilizing humectant.

Various other substances may be included in the oral preparations of this invention, such as bleaching agents, preservatives, silicones, chlorophyll compounds and/or ammoniated substances such as urea, ammonium hydrogen phosphate and mixtures thereof. These adjuvants, where present, are included in the formulations in amounts that do not significantly adversely affect the desired properties and characteristics.

Any suitable flavoring agents and sweeteners may also be used. Examples of suitable flavoring agents are aromatic oils such as spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, oregano, cinnamon, lemon and orange, and methyl salicylate. Suitable sweeteners include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP (aspartyl phenylalanine methyl ester), saccharin and the like. Suitably, the flavoring agent and sweetener may, individually or together, constitute from about 0.1 to 5% or more of the formulation.

The compositions of this invention may also be included in candies or chewing gum or other products, for example, by mixing with a warm gum base or coating the outer surface of a chew base, examples of which are jelutong, rubber latex, vinylite resin, and so on, as desired. with traditional plasticizers or softeners, sugar or other sweeteners such as glucose, sorbitol and the like. The composition of the invention may be a two-phase composition, where each phase allows the components to be released over different periods of time. For example, when used, the two-phase composition can release tin-associated stabilized ACP and/or tin-associated stabilized ACFP, preferably CPP-ACP/SnF ₂ and/or CPPACFP/SnF ₂ from the first phase at a greater rate than the compound capable of increasing or maintain the pH of the solution from the second phase. Preferably, the biphasic composition is a biphasic chewing gum.

An alternative composition may be a composition providing a phosphopeptide-stabilized ACP or ACFP and a tin compound, which then form tin-associated phosphopeptide-stabilized ACP or ACFP complexes in situ, for example, in the oral cavity. A typical composition may be a chewing gum containing a stabilized ACP or ACFP in the gum and a tin compound in the core.

The compositions of the invention 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 tin-associated PP-stabilized ACP or ACFP complexes of the invention are essentially the only active components of such a composition. For example, a composition may be used in the form of a cream containing: water; glycerol; complex according to the invention; D-sorbitol; silica; sodium carboxymethylcellulose (CMC-Na); propylene glycol; titanium dioxide; xylitol; phosphoric acid; guar gum; zinc oxide; sodium saccharin; ethyl nhydroxybenzoate; magnesium oxide; butyl n-hydroxybenzoate; and propyl n-hydroxybenzoate.

The invention also includes the composition described above provided together with instructions for its use for the treatment or prevention of dentin hypersensitivity.

- 13 043823

In another embodiment, the compositions of the invention as described herein do not contain a phosphate buffer and/or a calcium chelator. For example, any dentifrice described herein may not contain a phosphate buffer and/or a calcium chelator.

In one embodiment of the present invention there is provided a composition for the treatment or prevention of dentin hypersensitivity containing the PP-stabilized ACP and/or ACFP complex of the invention, without containing a phosphate buffer and/or a calcium chelator.

In another embodiment, the compositions of the invention as described herein contain no viscosity regulator or contain a viscosity regulator in an amount of from 0.5 to 50%.

In another embodiment, the compositions of the invention as described herein contain no sodium carboxymethylcellulose or contain from 0.01 to 10% sodium carboxymethylcellulose with an esterification degree of from 0.7 to 1.0.

In one embodiment, the active components of the composition consist primarily of the tin-associated PP-stabilized ACP or ACFP complexes of the invention.

In another aspect, a method of treating or preventing dentin hypersensitivity is provided, comprising the steps of administering a composition comprising a tin-associated PP stabilized complex of ACP and/or ACFP to the teeth of a subject. Local administration of the complex is preferable. The method preferably involves introducing the complex into compositions as described above.

Another aspect provides the use of the tin-associated PP-stabilized ACP and/or ACFP complex of the invention in the preparation of a composition for the treatment and/or prevention of dental hypersensitivity.

Another aspect provides the use of the tin-associated PP-stabilized ACP and/or ACFP complex of the invention in the preparation of a composition for mineralizing the surface or subsurface layer of a tooth.

According to another aspect of the invention, there is provided a composition for filling teeth, containing a filling material with the addition of a tin-associated PP-stabilized ACP and/or ACFP complex according to the invention. The basis of the filling material can be glass ionomer cement, composite material or any other compatible filling material. Glass ionomer cement is preferred. It is preferable that the amount of tin-associated PP-stabilized ACP or ACFP complexes included in the filling material is 0.01-80, preferably 1-5% by weight. The filling material of this invention containing the above agents can be prepared and used in various forms useful in dental practice. The restorative material of this embodiment may further contain other ions, for example, antibacterial ions Zn ²⁺ , Ag ⁺ and so on, or other additional ingredients, depending on the type and shape of the particular restorative material. Preferably, the pH of the filling material of this embodiment is 2-10, more preferably 5-9, and even more preferably 5-7. Preferably, the pH of the restorative material containing the tin-associated stabilized ACP or ACFP complex is in the range of about 2 to 10, more preferably about 5 to 9, and even more preferably from about 5 to 7.

It should be understood that although this description relates to specific embodiments of the invention in humans, it may also be used for veterinary purposes. Thus, in all aspects, the invention is applicable to farm animals such as cattle, sheep, horses and poultry, to domestic animals such as cats and dogs, and to animals kept in zoos.

One example of a mineralizing composition contains the following (in order of decreasing proportion): water;

glycerol;

CPP-ACP/SnF2;

D-sorbitol;

silica;

sodium carboxymethylcellulose (CMC-Na);

propylene glycol;

titanium dioxide;

xylitol;

phosphoric acid;

guar gum;

zinc oxide;

sodium saccharin;

ethyl n-hydroxybenzoate;

magnesium oxide;

butyl n-hydroxybenzoate;

Propyl-n-hydroxybenzoate.

- 14 043823

The invention will now be described with reference to the following non-limiting examples.

Example 1.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) was purchased from Cadbury Enterprises Pte Ltd under the trade name Recaldent™. Using CPP-ACP and SnF2, solutions were prepared to prepare each of the following tin-associated PP-stabilized ACP complexes:

0.4% CPP-ACP + 220 ppm F in the form of SnF2 (Sn:CPP molar ratio 8.6:1);

2% CPP-ACP + 500 ppm F in the form of SnF ₂ (molar ratio Sn:CPP 4:1); And

5% CPP-ACP + 500 ppm F in the form of SnF ₂ (molar ratio Sn:CPP 1.6:1).

Specifically, CPP-ACP/SnF ₂ complexes were prepared by first adding CPP-ACP to distilled/deionized water followed by slow addition of SnF 2 (as a solid or concentrated solution). If pH adjustment is necessary, it can be adjusted by adding 1 M HCl or 1 M NaOH to maintain a pH of 4.0-7.3. The pH was not allowed to rise above 7.3. There was no need for pH adjustment when using 5% CPP-ACP. The total volume of acid/base added was less than 1% of the volume of the CPP-ACP/SnF ₂ solution. The final solution contained tin-associated PP-stabilized ACP complexes.

Example 2.

The following describes the protocol for the analysis of ions in a solution of CPP-stabilized ACP/SnF ₂ . Samples for analysis of the total content of ions (strongly and loosely bound) and the content of loosely bound ions were obtained as follows.

Total content of ions (strongly and loosely bound). You can take one ml of any of the solutions (2)-(4) shown in example 3, place it in 19 ml of 1 M HNO3 and incubate at room temperature and constant stirring with slow turning for 24 hours (20 rpm). The mixture is centrifuged at 1000 g for 15 minutes at room temperature. The supernatant is analyzed for calcium, tin, phosphate and fluoride.

Content of loosely bound ions. Take a sample of the same solution that was used for the total ion analysis described above and place it in a centrifuge tube with a molecular weight cutoff (MWCO) of 1000 and centrifuge it at 3000 g for 1 hour at room temperature to obtain a sufficient amount filtrate (less than 10% of the total sample volume, so as not to disturb the equilibrium) for analysis by atomic absorption spectrophotometry (AAS) and ion chromatography (IC). The filtrates were then analyzed to determine the content of loosely bound ions.

The total and loosely bound calcium, tin, phosphate and fluoride contents in solution are determined by ion chromatography (for fluoride and phosphate) and atomic absorption spectrophotometry (for calcium and tin).

The content of ions tightly bound to CPP (colloidal retentate) is calculated from the difference between the total content and the content of loosely bound ions (as explained above).

Example 3.

Self-assembly of the SnF ₂ /CPP-ACP nanofilament network on dentin at a Sn/CPP molar ratio of 1.6 results in the most effective closure of open dentinal tubules and can therefore most effectively reduce dentin hypersensitivity.

According to the hydrodynamic theory of dental hypersensitivity, restricting the movement of fluid along the dentinal tubules can lead to clinical reduction of hypersensitivity. In this example, the inventor demonstrates the synergistic effect of the combination of CPP-ACP and SnF ₂ at a Sn(II)/CPP molar ratio of 1.6 on the closure of tubules on the dentin surface using scanning electron microscopy (SEM).

Sections of extracted human third molars were prepared to produce coronal discs approximately 1 mm thick. The discs were coated with acid-resistant nail varnish, leaving the central dentin area exposed, and immersed in 15% EDTA for 2 minutes to remove the smear layer. The discs were randomly assigned to one of four groups and subjected to the following treatment for 20 minutes:

1) treatment with double distilled water (DDW);

2) 0.4% CPP-ACP + 220 ppm F in the form of SnF ₂ (Sn:CPP molar ratio 8.6:1);

3) 2% CPP-ACP + 500 ppm. F in the form of SnF ₂ (molar ratio Sn:CPP 4:1); And

4) 5% CPP-ACP + 500 ppm. F in the form of SnF ₂ (molar ratio Sn:CPP 1.6:1). The discs were then dehydrated and gold sputter coated for subsequent SEM analysis.

In the SEM images shown in FIGS. 2-6, it is shown that dentin subjected to treatments (2)-(4) exhibited surface deposition and varying degrees of dentinal tubule closure. The negative control, DDW treatment, demonstrated smooth dentin surfaces with open tubules (Figure 1). Combined solution 500 ppm F SnF ₂ and 5% CPP-ACP (Sn/CPP molar ratio 1.6) interacted with the dentin surface, resulting in a unique surface coverage pattern of a self-assembled nanofilament network on the dentin surface,

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Claims (13)

closing the tubules (Fig. 2-4). These nanofilaments are believed to be CPP cross-linked with Sn, releasing its calcium, phosphate and fluoride ions to the surface to form nanofilaments. Only a solution with a Sn/CPP molar ratio of 1.6 resulted in the formation of cross-linked nanofilaments, indicating that a molar ratio equal to or greater than 1 mol but less than 4 mol Sn(II) per mole of CPP is critical for the formation of cross-linked nanofilaments. cross-linked nanofilaments and a greater reduction in dentin hypersensitivity. Treatment of exposed dentin with a solution with a Sn:CPP molar ratio of 8.6:1 resulted in only partial closure of the dentinal tubules (Fig. 6). Similarly, treatment of exposed dentin with a 4:1 Sn:CPP molar solution provided only partial closure (Figure 5). It should be understood that the invention as disclosed and defined herein covers all alternative combinations of two or more individual features indicated or apparent from the text or graphics presented. All of these different combinations constitute various alternative aspects of the invention. CLAIM 1. A tin-associated complex of amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP), stabilized by phosphopeptide (PP), containing tin ions from about 1.4 to about 1.8 moles of tin per mole of PP. 2. The complex according to claim 1, wherein the phosphopeptide is a casein phosphopeptide. 3. A composition containing the complex according to claim 1 or 2 and a pharmaceutically acceptable carrier, diluent or excipient. 4. The composition according to claim 3, selected from dentifrices, including toothpastes, toothpowders and liquid dentifrices, mouthwashes, mouthwashes, oral sprays, dental varnish, dental cement, lozenges, chewing gum, dental pastes, gum massage creams, mouthwash tablets, dairy products and other food products. 5. A method of treating or preventing dentin sensitivity in a subject in need thereof having exposed dentin, comprising administering a tin-associated phosphopeptide (PP) stabilized ACP or ACFP complex with a tin ion content equal to or greater than 1 mole of tin per mole of PP, but less than 4 mol tin per mole PP, or a composition containing a tin-associated phosphopeptide (PP)-stabilized ACP or ACFP complex with a tin ion content equal to or greater than 1 mol tin per mole PP but less than 4 mol tin per mole PP, and pharmaceutical an acceptable carrier, diluent or excipient that provides treatment or prevention of dentin sensitivity in an individual. 6. The method according to claim 5, where the open dentin contains dentinal tubules, the diameter of the holes of which exceeds 1.0 μm. 7. The method according to claim 5 or 6, wherein the phosphopeptide is a casein phosphopeptide. 8. The method according to any one of claims 5 to 7, where the composition is a composition selected from a dentifrice, including toothpastes, toothpowders and liquid dentifrices, mouthwashes, mouthwashes, sprays for oral cavity, dental varnish, dental cement, lozenges, chewing gums, dental pastes, gum massage creams, mouthwash tablets, dairy products and other food products. 9. The use of a tin-associated complex of ACP or ACFP, stabilized with phosphopeptide (PP), with a tin ion content equal to or greater than 1 mol tin per mole PP, but less than 4 mol tin per mole PP, in the manufacture of a medicinal product for the treatment or prevention of hypertension dentin sensitivity in an individual with exposed dentin. 10. Application according to claim 9, where the exposed dentin contains dentinal tubules, the diameter of the holes of which exceeds 1.0 μm. 11. Use according to claim 9 or 10, wherein the phosphopeptide is a casein phosphopeptide. 12. The method according to any one of claims 5-8, where the complex or composition is administered directly to the area of the oral cavity in need of treatment. 13. The method according to any one of claims 5 to 8, wherein the area of the oral cavity in need of treatment is brought into contact for a period of about 10 to 60, or about 20 to 30, or about 20 minutes. -