AU2009202295A1 - Calcium Phosphate Nanoclusters - Google Patents

Description

WO 01/44106 PCT/CB00/04827 I CALCIUM PHOSPHATE NAVOCLUSTERS AND TEIR APPLICATIONS 2 3 The present inventicn relates to the field of calcium 4 phosphate nanoclusters and their applications in high 5 calcium content nutritional products, nutraceuticals and 6 pharmacological preparations for use in treatment of 7 pathological calcification. 8 9 It is important for mammals to have a high intake of 10 dietary calcium. When this is not achieved in humans, II bone diseases such as rickets or osteoporosis may result. 12 There is a market for high calcium content nutritional 13 and nutraceutical preparations; for example, calcium 14 tablets for prevention of osteoporosis and 15 sports/nutraceutical calcium containing drinks. Clearly, 16 milk contains a high concentration of calcium and many of 17 these drinks deliberately aim to provide calcium without is the fat etc. associated with milk. Many of these drinks 19 contain calcium gluconate, being a salt of calcium with a 20 reasonably high solubility, allowing the drink to have 21 the same calcium content as milk but with a lower 2- phosphate concentration. However, these are limited in 23 their maximum calcium concentration and also have a sweet SUBSTITUTE SHEET (RULE 26) WO 0144106 PCT/GBO0J4827 2 1 taste (and some calorie content) due to the gluconate 2 which is not always appropriate to the application. 4 It is a first aim of the present invention to provide a 5 method for achieving drinks, solid foods, supplements and 6 the like containing a higher concentration of calcium 7 than is possible with calcium gluconate. The higher the & concentration that can be achieved, the lower the volume 9 that must be drunk to enable a consumer to gain their 10 reconanended daily amount of calcium. A linked aim is to 11 provide stable materials that can be easily stored, 12 preferably for long period of time. A further linked aim 13 is to provide heat-sterilizable high calcium materials. 14 15 A further linked aim is to provide a soluble formulation 16 of essential trace elements in a high calcium solution as 17 an aid to the absorption of otherwise insoluble 18 micronutrients. 19 20 In the body, there are pathologies associated with the 21 precipitation of calcium. For example, pathological 22 calcification in the mammary gland is a problematic 23 disease in which milk stones corporaa amylacea) form. An 24 additional major aim of the present invention is to 25 provide a preparation for the treatment of pathological 26 calcification. This might be achieved by binding the 27 nanoclusters to a suitable ion-permeable matrix, for 28 example gelatin gel. A gel of caseins might also be 29 used. This would have the advantage of not having other 30 potentially allergenic material in its structure. 31 32 Calcium phosphate nanoclusters would also be applicable 33 to dental care products such as mouth washes and 34 toothpastes. Exposure of teeth to calcium phosphate SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GBOO/04827 I nanociusters will inhibit or prevent the demineralisatio. 2 of teeth and application of a phosphopeptide containing 3 gel or other suitable solid containing phase to the teeth 4 could inhibit calcification of dental plaque or even S reverse it. A further application would be in the 6 prevention of calcification of medical prostheses such as 7 urinary catheters and heart valves since pathological 8 calcification is a major reason for the failure of these 9 devices. In this method for preventing pathological 10 calcification there would be formed on the surface an if equilibrium nanocluster like- structure, which can be 12 reversed at will, rather than irreversible precipitation 13 of a calcium salt. 14 15 In mammals, nature has already addressed the problems of 16 calcium concentration in the form of milk. Milk contains 17 a high concentration of calcium and phosphate as a 18 foodstuff for growing young. Mammalian milk has been 19 found to contain casein micelles which contain calcium, 20 phosphate and casein. It has been hypothesized that a 21 function of these micelles is to allow milk to have a 22 high calcium and phosphate concentration without causing 23 pathological calcification in normal conditions. 24 25 In recent years, artificial particles containing high 26 concentrations of calcium and phosphate have been 27 disclosed. [ Carl HOLT et al., Biochem J., 314, 1035 28 1039 (1996) ; Carl HOLT et al., Eur. J. Biochemr 252, 73 29 78 (1998) ; Carl HOLT et al., Advanced Dairy Chemistry, 30 Vol. 3: Lactose, Water, Salts and Vitamins (P. F. Fox, 31 Ed.) ] These are known as Calcium Phosphate 32 nanoclusters. 33 SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCTIGBOM04827 4 1 Calcium phosphate nanoclusters have been made from 2 complex salt mixtures; for example, 4 37mM Ca(N0 3 ), 6mM Mg(NO3)2, 3-6mM KNO 3 , 25mM KR 2

PO

4 , 5mM 5 K 2 HP0 4 , -1.54mM NaN3 ( as a preservative ) [ Holt et al., J 6 1998] 7 8 or 9 to 30mM Ca(NO3), 4mM Mg(NO 3 )2, 1OmM tripotassium citrate, i 20mM KH 2

PO

4 , 26mM KN03, 1.5mM NaN3 ( as a preservative ) [ 12 Holt et al., 1996 13 14 These solutions also contained a pure single is phosphopeptide, S-casein 4P(fl-25) I Holt et al., 1996 ], 16 or p-casein 5P(fl-42) [ Holt et al., 1998 1. 17 is Both of these solutions were initially at pH5.5. The pH 19 has to be raised to form calcium phosphate nanoclusters. 20 A problem perceived in these papers was that if the salt 21 solutions were mixed crudely with e.g. a higher pH 22 solution, there would be crude precipitation of calcium 23 phosphate, spoiling the formation of nanoclusters. 24 Furthermore, variation throughout two solutions being 25 mixed would lead to nonuniform nanoclusters, reducing 26 their homogeneity. This problem was solved in these 27 papers by generating ammonia homogeneously in solution 28 from the breakdown of urea by jack bean urease. 29 30 At the present date, the publicly understood theory of 31 nanocluster formation is that calcium phosphate 32 precipitates from a supersaturated solution but does so 33 by first forming nuclei tha. grow to form the macroscopic 34 phase. The action of the phosphopeptide is perceived to SUBSTITUTE STREET (RULE 26) WO 01/44106 PCT/GBO0/04827 5 I be one in which it coats the nuclei in a protective layer 2 which slows down their growth into the precipitate 3 (kinetic stabilization theory). This theory is clearly 4 stated in the latest published work [Holt, C., Timmin_ 5 P. A., Errington, N. & Leaver, J. (1998). A core-sheli. 6 model of calcium phosphate nanoclusters derived from 7 sedimentation equilibrium and small angle X-ray and 9 neutron scattering measurements European Journal of 9 Biochemistry 252, 73-78] is extensively described in a 10 review article [(Holt, C. (1997) The milk salts and their 11 interaction with casein. In: Advanced Dairy Chemistry, 12 Vol. 3: Lactose, Water, Salts and Vitamins (P. F. Fox, 13 Ed.), Chapman and Hall, London, pp- 233-254)}. Thus, 14 nanoclusters are seen as intrinsically unstable particles 15 that would, given enough time, themselves precipitate. 16 Hence, magnesium and citrate, which are known to be 17 inhibitors of the rate of precipitation are thought to 12 help the more powerful phosphopeptide to form stable 19 intermediate nanoclusters and so are considered 20 essential. Magnesium and citrate present in solution 21 were found to be incorporated into the resulting 22 nanoclusrers (Holt et al. 1996). The urea/urease method 23 has been used to raise the pH as the gentlest possible 24 method of doing so. 25 26 According to the kinetic stabilization theory, simple 27 mixing together of strong base with an acidic solution of 28 calcium, phosphate and phosphopeptide would givg rise to 29 a product which depended on the exact conditions of 30 stirring and the transient concentration gradients of 31 mixing and which would therefore be difficult to 32 reproduce or do rapidly. Moreover, in the presence of 33 even tiny amounts of the thermodynamically more stable SUBSTITUTE SHEET (RULE 26) WO 01144106 PCTIGBOO/04827 6 I precipitated phase, the precipitate would grow at the 2 expense of the nanoclusters. 4 Lastly, currently accepted theory suggests that a mixture 5 of phosphopeptides, such as the ones obtained from whole 6 casein would be ineffective since some of the 7 phosphopeptides would be capable of bridging between two 8 nanocluster particles to create a network or gel while 9 others contained fewer than the requisite number of 10 phosphorylated residues. Thus it is thought that 11 nanocluster formation would only occur under carefully 12 controlled conditions which eliminated concentration and 13 pH gradients. Hence the urea/urease method is 14 considered essential to forming stable nanocluster 15 solutions and it is also considered imperative to use a 16 pure phosphopeptide. 17 18 There is described herein developments which show that 19 this generally accepted model is incorrect. As a result, 20 a simpler and substantially more commercially viable 21 method of preparing calcium phosphate nanoclusters has 22 been developed and is described herein. .Furthermore, 23 nanoclusters of a new composition are also disclosed. 24 25 In this document, a centre of phosphorylation is a region 26 of a peptide or protein containing 3 or more 27 phosphorylated residues in a short sequence. 28 29 According to a first aspect of the present invention 30 there is provided a method of making calcium phosphate 31 nanoclusters comprising the preparation of a nanocluster 32 forming solution, a nanocluster forming solution 33 containing appropriate concentrations of calcium ions, 34 phosphate ions and multiply phosphorylated phosphopeptide SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GB0/04827 7 1 molecules and having an appropriate pH for formation of 2 calcium phosphate nanoclusters, the method being 3 characterized in that the calc.-m phosphate nanocluster 4 forming solution is made by mixing together two solutis 5 neither of which is itself a nanocluster forming solutj~on 6 but which contain components such that, when they are 7 combined, they form a calcium phosphate nanocluster 8 forming solution. 9 10 Preferably, a phosphopeptide molecule has a Centre of 11 phosphorylation. 12 13 Preferably, a centre of phosphcrylation has at least 3 14 phosphorylated residues. 15 16 More preferably, a centre of phosphorylation has at least 17 3 phosphorylated residues close together in the multiply is phosphorylated peptide. 19 20 Most preferably, a centre of phosphorylation has at least 21 3 phosphorylated residues in a series of 6 consecutive 22 residues in the primary structure of the phosphopeptide 23 molecule. 24 25 Preferably, a phosphopeptide molecule contains few or no 26 hydrophobic regions. 27 29 Preferably also, a phosphopeptide molecule contains only 29 one centre of phosphorylation. 30 31 A nanocluster may have a plurality of different types of 32 phosphopeptide molecule. 33 SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCTIGBO/04827 8 I Typically, the phosphopeptide molecules will be a 2 mixture. 3 4 The mixture of phosphopeptide molecules may be an 3 enzymatic digest of a crude casein preparation. 6 7 Typically, the thermodynamic activities of calcium and 8 phosphate ions in solution formed from the two solutions 9 combined are large enough to exceed a solubility-product 10 type relationship such as that found in milk: K1 {Ca 2 +)(p O _ 0

.

2 {HPO ~)0.7; 1.6.10-7 5 10-6 M 1 '. 12 Where the curly brackets denore activity. 13 14 Most preferably, the nanocluster forming solution has 15 [Cat) / PP] s 3 ; [Pi,t] = (0.875±0.125), [ PP) - 1.67 t 16 0.25 ; where [PP) is the concentration of the 17 phosphopeptide in grams per litre, [Pil is the total is millimolar concentration of inorganic phosphate and (Ca] 19 is the total millimolar calcium concentration. 20 21 Preferably also, the nanocluster forming solution does 22 not contain significant amounts (greater than 0. ImM) of 23 other calcium chelating agents. 24 25 Preferably, if a calcium chelating agent such as nitrate 26 is included there must be an increased amount of calcium 27 allowed in the formulation. 28 29. The nanocluster forming solution may contain no 30 magnesium. 31 32 A dispersing agent may be added. 33 34 A preservative may also be added. SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCF7GB00/04827 9 2 The resulting nanocluster solution may be sterilized. 4 The resulting nanocluster solution may be freeze dried .

6 According to a second aspect of the present invention 7 there are calcium phosphate nanoclusters wherein each 8 nanocluster comprises calcium, phosphate and multiply 9 phosphorylated phosphopeptide molecules, provided that no 10 more than 80% of the phosphopeptide molecules are p il casein 4P (fl-25) and provided also that no more than 60% 12 of the phosphopeptide molecules are 0-casein 5P (fl-42). 13 14 A calcium phosphate nanocluster formed from a solution 15 containing the $-casein phosphopeptide residues 1-25 has 16 the empirical formula [a,, (HPOl )..(PO3-). 5 5

(H

2 O),. I [Ca 4 . SerP, - Casein) 17 is wherein SerP 4 -Casein is the phosphopeptide. These 19 particles are thermodynamically stable in the pH range 20 6.0-7.2 and have a solubility product, K,, given by: KS= {Ca2. )-o {Po O r,-*o3r K, = 8. 671(± 0.600).10-M'- 76 21 22 Preferably, the nanoclusters have an empirical formula in 23 the range: [Ca(HPO ) 0 3(Po 4

")-(H

2 O)xls..Caz-SerP-peptde] 24 where(y23)and the sum of charges of the ions within both 25 square brackets is approximately zero and where Ca 24 SerP , SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GBOO/04827 10 I peptide is the calcium salt of a multiply phosphorylated 2 phosphopeptide molecule. 4 Preferably, a phosphopeptide molecule has a centre of 5 phosphorylation. 6 7 Preferably, a centre of phosphorylation has at least 3. 8 phosphorylated residues. 9 to More preferably, a centre of phosphorylation has at least 11 3 phosphorylated residues close together in the multiply 12 phosphorylated peptide. 13 14 Most preferably, a centre of phosphorylation has at least 15 3 phosphorylated residues in a series of 6 consecutive 16 residues in the primary structure of the phosphopeptide 17 molecule. 18 19 Preferably, a phosphopeptide molecule contains few or no 20 hydrophobic regions. 21 22 Preferably also, a phosphopeptide molecule contains only 23 one centre of phosphorylation. 24 25 A nanocluster may have a plurality of different types of 26 phosphopeptide molecule. 27 28 A plurality of different types of phosphopeptide molecule 29 may be selected from phosphopeptide molecules derived 30 'flom an enzymatic digest of a crude casein preparation. 31 32 Typically, the nanocluster will comprise 10-200 of the 33 approximate empirical formula units. 34 SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCTGBOO/04827 11 i Preferably, the nanocluster does not have significant 2 amounts of magnesium therein. 3 4 Preferably also, the nanocluster does not have 5 significant amounts of citrate therein. 6 7 A dispersing agent may be included. 8 9 A preservative may also be included. 10 11 The nanoclusters may be sterilized. 12 13 According to a third aspect of the present invention 14 there are provided nanoclusters obtainable by the method 15 of first aspect. 16 17 According to a fourth aspect of the present invention is there is provided a calcium containing food or beverage 19 comprising nanoclusters according to the second or third 20 aspect. 21 22 According to a fifth aspect of the present invention 23 there is provided a pharmaceutical composition for the 24 inhibition or prevention of tooth demineralisation, the 25 composition comprising nanoclusters according to the 26 second or third aspect. 27 28 According to a sixth aspect of the present invstion, 29 there is provided a pharmaceutical composition for the 30 treatment or prevention of pathological calcification, 31 the composition comprising phosphopeptides according to 32 the second or third aspect and a pharmaceutically 33 acceptable carrier. 34 SUBSTITUTE SHFET (RULE 26) WO 01/44106 PCr/GBOO/04827 12 I According to a seventh aspect of the present invention 2 there is provided a pharmaceutical; composition for a 3 soluble mixture of essential trace elements. 4 3 The pharmaceutically acceptable carrier may comprise an 6 ion permdable matrix. 7 8 Preferably, the ion permeable matrix is a gel. 9 10 The ion permeable matrix may be a casein based gel. II 12 The present invention will now be described with 13 reference to the following Figures in which: 14 Is Figures la and lb show reversed phase high pressure 16 liquid chromatograms following a modification of the 17 procedure of Ellegird et al. (1999) of (la) the Is retentate and ultrafiltrate of a calcium phosphate 19 nanocluster solution, prepared by a method similar 20 to A4 and (lb) an optimised subtraction of the 21 ultrafiltrate from the retentate. 23 Examples of a method for making calcium phosphate 24 nanoclusters are described in depth below. In general, 25 the method consists of a defined sequence of operations 26 involving first the dissolution of the phosphopeptide 27 powder in a volume of water, followed by the addition of 28 known volumes of stock solutions of salts, including 29 phosphate salts (e-g. sodium dihydrogen phosphate and/or 30 '-sodium hydrogen phosphate and or trisodium phosphate), 31 and, depending on the final pH to be attained, a volume 32 may be added of either an acid or an alkali. It is 33 preferred that, in the final stage, a volume of a stock SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GBOO/04827 13 I solution of a calcium salt is added with stirring to 2 achieve the final composition and pH. 4 The order in which the stock solutions are added can Be' 5 varied so that the final stage could be either a final' 6 adjustment of pH with an alkali or a final addition of 7 the calcium solution or one of the phosphate salts but in s all cases it is preferred that the addition is made 9 slowly and with stirring to minimise the turbidity of the 10 solution. In the presence of sufficient phosphopeptide, it larger nanoclusters formed during mixing will decrease in 12 size slowly to an equilibrium value, and even 13 precipitated calcium phosphate will spontaneously 14 redisperse. 15 16 The new method of making nanoclusters differs from the . 17 previously published method in three essential respects. 18 First it uses different phosphopeptides or 19 phosphoproteins or a mixture of phosphopeptides rather 20 than a single peptide. Second, it uses a different 21 procedure for achieving the final pH which was only 22 considered possible after further undisclosed research 23 had been completed. The method differs in a third, 24 respect in that neither magnesium nor citrate salts were 25 found to be needed and were eliminated for normal 26 purposes. 27 21 This method has been developed from never prevIously 29 disclosed research which contradicts the current theory 30 of nanocluster formation. We have shown that the 31 nanoclusters are not kinetically stabilized but in fact 32 have thermodynamic stability. In a first experiment, a 33 nanocluster solution was taken to pH1>8 where it 34 precipitated, as thought, into a more thermodynamically SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GB00/04827 14 I stable state. However, when the suspension of the 2 precipitate was readjusted to neutral pH, the precipitate 3 spontaneously reformed the nanoclusters. In a second, 4 even more convincing, experiment, phosphopeptide was 5 added to a fresh calcium phosphate precipitate wherein 6 the pregipitate.was seen to disappear slowly even though 7 the condition of supersaturation was not changed. In 8 other words, the nanoclusters are more thermodynamically 9 stable than the precipitated phase. There is no known. 1o precedent for this result. i 12 These experiments lead to a new theory which states that 13 the nanoclusters are thermodynamically stable entities 14 and we need not be so concerned about the kinetic, non 15 equilibrium aspects of their formation since they will 16 achieve the core-shell nanocluster structure on standing 17 even if there is some initial precipitation. We 18 therefore experimented with simple mixing procedures that 19 gave a minimum amount of precipitation and found that 20 they worked. The surprising discovery that simple 21 mixing can be used to form nanoclusters, in flat 2 contravention of the currently accepted theory, is of 23 great benefit in that it reduces the complexity of the 24 manufacturing process. 25 26 The method herein disclosed preferably uses a 27 phosphopeptide mixture. Again, currently accepted theory 28 described above indicates that if a mixture of 29 phosphopeptides were used, some would be capable of 30 ''bridging between two nanocluster particles, giving a 31 network or gel. Given accepted theory at the present 32 time, it is therefore surprising that a mixture may be 33 used. It is much easier to manufacture a phosphopeptide 34 mixture than it is to manufacture a pure phosphopeptide. SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GB00/04827 2 Furthermore, the method disclosed herein eliminates 3 magnesium and citrate. As discussed above, magnesium and 4 citrate were though to help the more powerful 5 phosphopeptide to form important stable intermediate 4 6 nanoclusters as part of the nanocluster formation 7 process. As magnesium and citrate are known inhibitors 8 of the rate of precipitation, the surprising discovery 9 that they may be removed is of great benefit. 10 11 With all these changes in method of preparation in 12 comparison with the prior Art and in contravention of 13 previously accepted theory, nanocluster-like particles 14 are still formed but they can have a different 15 composition, namely that different phosphopeptides are 16 present in the shell. As a general rule, only 17 phosphopeptides containing three or more phosphorylated 18 residues within a short sequence of the primary structure 19 are capable of forming (part of) the shell and in a 20 casein phosphopeptide mixture, all peptides satisfying 21 this criterion are bound in the nanoclusters to some 22 degree. 23 24 The resulting calcium phosphate nanoclusters are 25 particles having a core-shell structure with the core 26 comprising largely a salt of calcium phosphate with a 27 radius of a few nanometers and a shell composed largely 28 of a phosphopeptide or a mixture of phosphopepeides or a 29 phosphoprotein or a mixture of phosphoproteins. 30 31 Other components that are not essential to the formation 32 of the nanocluster can become incorporated, either by 33 design or accident. For example, magnesium ions and 34 citrate ions can become bound to the nanoclusters if SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GBOO/04827 16 1 present in the solution. If P-casein is used as the 2 phosphoprotein then a dispersing agent such as K-casein 3 can be used to prevent or reduce adherence of the 4 nanoclusters to form larger macroclusters or colloidal 5 aggregates. 6 7 Calcium phosphate nanoclusters can form and be stable at 8 about neutral pH but can form a gelatinous phase at pH 8 9 or greater. . Nanoclusters can form if the solution is 10 saturated with respect to calcium phosphate, as defined 11 by a solubility product relationship which takes the 12 approximate form in milk of: 13 14 K,= (Ca2+){pO3 ) 0

.

2 (HPO 20.7; 1.6.'I K 104 M". (Equaion ) 16 Where Ks is a constant for a given type of nanocluster, 17 (Ca 2 +) is the activity of calcium ions, {POt ) is the 18 activity of the phosphate trianion and (HPO1) is the 19 activity of the phosphate dianion. 20 21 The Calcium phosphate nanoclusters produced have an 22 approximate empirical formula: [Ca.

4 .. (H po-X , (,PO - .(HO), .] [Ca 4 .,SerP 4 - Casein] 23 24 where Ca 4

.

7 SerP 4 -peptide is a calcium salt of a 25 phosphopeptide containing preferably at least three and 26 usually four phosphorylated residues close together in 27 t;he primary structure. However, the composition can 28' -ry depending on the rate of pH change in the final 29 stage of formation, the composition of the salt solution 30 and the amount and nature of the phosphopeptide. 31 32 The range of possible concentrations is thought to be: SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCI'/GB0O/04837 17 2 [C(HPO ) 0 (PO~)0 3

(HZO).S

1

,

5 .[Ca, 4 SerP,-peptide] (Equation 2a) 3 where (ya3) and the sum of charges within both square 4 brackets is approximately zero. 5 6 An actual nanocluster will comprise some, usually small, 7 number of multiples of the empirical formula, for example 8 49 ± 4 in the standard nanocluster preparation of Holt et 9 al. (1998). 10 11 Notwithstanding the above, the constraints on the 12 composition of a solution able to form stable calcium 13 phosphate nanoclusters can be represented by the 14 following approximate empirical relations which apply to 15 a commercial phosphopeptide preparation (PP) derived from 16 a tryptic digest of whole casein in a solution of calcium 17 and phosphate salts but containing no more than minor 18 amounts of other strong calcium chelating agents such as 19 citrate. 20 21 (Cal / [PP] : 1.5. (Equation 3) 22 [Pij] (0. 875±O.125) . [ PP] - 1. 67 ± 0.25 (Equation 4) 23 24 where (PP] is the concentration of the phosphopeptide in 25 grams per litre, [Pi,t] is the total millimolar 26 concentration of inorganic phosphate and (Cat] is the 27 total millimolar calcium concentration. 28 29 A range of phosphopeptides may be used, as indicated in 30 the following investigation: 31 SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GB00/04827 18 I Nanocluster formation was investigated with the following 2 phosphoproteins, phosphoprotein mixtures, phosphopeptides 3 and phosphopeptide mixtures: 4 5 1. Whole casein 6 2. Mixtures of A- and K-caseins 7 3. p-casein SP 8 4. A commercial phosphopeptide mixture supplied by MD 9 foods and derived from casein, containing a large 10 number of phosphopeptides as described by Ellegird et 11 al.. (1999). 12 5. A phosphopeptide mixture derived from a digestion of 13 whole casein with protease XIV from Streptomyces 14 griseus, and papain type IV containing 'as--casein 2P Is (f46-51),. asl-casein 4P (f61-70), 0-casein 4P (fll-21), 16 us-casein 3P (f5-12), as2-casein 4P (f49-61) and us2 17 casein 2P (fl26-133). is 6. p-casein phosphopeptides 4P (fl-25) or 4P (f2-25) or 5P 19 (fl-42) 20 7. Cyanogen bromide cleavage fragments u 5 --casein 2P (fl 21 54) or asi-casein 6P (f61-123). 22 1. Tryptic phosphopeptides asl-casein 1P (fl04-119) or ast 23 casein 2P (f43-58) or ae 5 I-casein SP (f59-79) or ast 24 casein 7P (f43-79) 2S 26 A test for nanocluster formation was applied in which the 27 standard conditions for the formation of nanoclusters 28 'rom the 0-casein phosphopeptide were modified to the 29 smallest extent possible and applied to the other 30 peptides or peptide mixtures. Absence of precipitation 31 was evidence of the formation of nanocluster-like 32 particles. Conditions were found with the SUBSTITUTE SHEET (RULE 26) WO 01/44106 PC/CB00/048Z7 19 I phosphopeptide mixes 4 and S and the phosphoproteins 1 2 and 2 where a stable clear Solution was formed but 3 3 underwent an unlimited aggregation. With 4, HPLC showed 4 that the less phosphorylated peptides were concentrate4 5 in the serum rather than being on the nanoclusters. ;6 '6 Formation of nanocluster-like particles by 4 was 7 confirmed experimentally by X-ray scattering 8 measurements. Nanoclusters were found with each of the 9 6 phosphopeptides and confirmed experimentally by X-ray 10 scattering measurements. The cyanogen bromide peptides n1 of 7 either failed to dissolve (oij-casein 2P (fl-54)) or 12 formed a precipitate in the test system (al-casein 6P 13 (f61-123). Nanoclusters were formed with either the 5? 14 or 7P fractions of 8 and confirmed experimentally by X 15 ray scattering measurements. No stable nanocluster 16 solution was formed with either the 1P or 2P peptide in 17 8. 19 In conclusion, peptides or proteins containing 3 or more 20 phosphorylated residues in a short sequence of the 21 primary structure are able to form calcium phosphate 22 nanocluster-like particles under favourable conditions. 23 A typical centre of phosphorylation in casein would have 24 4 phosphorylated residues in the space of 5 or 6 amino 25 acids. 26 27 Peptides should contain few if any hydrophobic regions to 28 avoid the use of another dispersing agent but sych an 29 agent can be used as needed. The peptides should 30 preferably contain only one centre of phosphorylation to 31 avoid cross linking although a small amount of such a 32 peptide in mixtures with other suitable peptides does not 33 cause serious problems. The peptide should preferably 34 be readily soluble. This new research indicates the SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GRBO/04827 20 I general principle which can be used to find other useful 2 phosphopeptides; for example, digests of a 5 5 -casein, 3 phosvitin and cz,-casein. 4 5 Equationg 1 and 2 together define within fairly close 6 limits the extent of formation of nanocluster particles 7 in solutions of calcium and phosphate salts, a background a electrolyte and a suitable phosphoprotein or 9 phosphopeptide. Significant modifications of the 10 composition of the solution by the inclusion for example ii of other, normally multivalent, anions or cations, 12 requires a modification to equations 1 and 2 to take 13 account of the strong interactions. 14 15 The upper limit of calcium concentration possible using 16 the calcium phosphate nanocluster system within the scope 17 of the present invention is not known. However, initial is experimentation has managed to prepare solutions up to 30 19 times more concentrated than cow's milk. It should be 20 possible to provide very high concentrations of Calcium, 21 e.g. IM compared to 0.008M in human milk. This allows 2- the preparation of nutraceutical drinks, foodstuffs or 23 additives for food and drink which allow a consumer to 24 gain a high calcium intake from only a small volume of 25 produce. 26 27 Example method 28' ,Te example method uses a commercial phosphopeptide 29 mixture from MD Foods (NaCPP) described by K.H. Ellegbrd, 30 C. Gammelgird-Larsen, E. S. Serensen & S. Fedosov. 31 Process scale chromatographic isolation, characterization 32 and identification of tryptic bioactive casein SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GB00/04827 21 I phosphopeptides, International Dairy Journal 9 639-652 2 (1999). 3 4 The sample was prepared using the following stock 5 solutions made up to the indicated molar concentration-. 6 with deionised water: 7 8 Table I Stock solutions used to form calcium phosphate nanoclusters Substance Concentraion (M) CaC 2 0.200 NaCI 1000 NaH!P0 4 0.200 NaJIPO 4 0.200 NaPO4 0.200 NaOH 1.000 NaNj (1%) 0.154 9 10 The sodium azide addition is for inhibition of bacterial ii growth and may be omitted or replaced by filter or steam 12 sterilization, as appropriate to the end use. Sodium 13 chloride is not important in nanocluster formation and is 14 present primarily to give a particular application iS related ionic strength, for example, to give a solution 16 with an ionic strength approximately that of baby milk. 17 Potassium salts may be used in place of the sodium salts 18 provided the solubility is great enough and the chloride 19 salts can be replaced by nitrates or any other equally 20 soluble salt. 21 22 The peptide is dissolved in a certain volume oftwater 23 calculated as the final volume less the sum of the 24 volumes of each of the stock solutions required to give 25 the final composition and less a volume contribution from 26 the peptide 'which is calculated from an assumed partial 27 specific volume of 0.7 ml g 4 . SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/CB427 22 I Method Al preparation of 3 =l of solution containing 30 2 mM Ca. 3 4 To 60 mg of the phosphopeptide powder, add 2.145 ml 5 deionised water with stirring until dissolved. Place a 6 calibratied glass pH electrode in the solution and stir, 7 using a magnetic follower at about 60 rpm while measuring 8 the pH after every addition of one of the above stock 9 solutions. All the additions are made over a period of 10 about 30 min. With the objective of obtaining a final 11 pH of 6.7±0.05 but without ever allowing the solution to 12 become more alkaline than 6.75, the order of addition is 13 as follows: 14 Table 2 Example sequence of additions Stock Solution Addition Cumulative Volume pH (pal) (ml) CaL 450 2.595 6.91 NaCI ISO 2.745 6.942 NaN, 30 2.775 6.995 NaHPO 4 20 2.795 6.764 Na 2

HPO

4 30 2.825 6.704 NaH2POj 10 2.835 6.653 NaHPO4 40 2.875 6.675 Na2HPO4 60 2.935 6.719 NaH2PO 10 2.945 6.671 NajMPO, 30 2.975 6.703 NaH3PO4 10 2.985 6.665 NaHP15 3.000 6.686 15 16 The final composition of the sample was as follows: 17 18 , able 3 -Final composition of the nanocluster solution Substance Concenwation, mM Ca 30 Total inorganic P 15 Na 76.66 Cl 110 SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCr/CB00/04827 Total oranic P' 19.61 Total peptide1 8.94 I ' 3.03%w/w Total P in the N&CPP 2 2 Mean pepde mass 2,237 Da 3 Method A2 Preparation of 3 ml of solution containing 30 4 mM Ca. 6 Add all the ingredients as single additions of the stock 7 solutions after first dissolving the phosphopeptide 8 powder in the water. All additions are made slowly by 9 micropipette with the pipette tip submerged. Although 10 the pH becomes more alkali:e than the target final pH, 11 this occurs in the absence of calcium and the addition of 12 the calcium chloride brings the pH down so turbidity does 13 not develop. 14 15 Table 4 Example additions to form the nanocluster solution Stock Solution Addition Cumulative Volume pH (pW (nil) H2O 2103 2.145 NaN, 30 2.175 NaCI 150 2.325 NaH 2

PO

4 50 2.375 7.239 NatHP0 4 175 2.550 17.471 Cach 450 3.000 1 6.710 16 Method A2 is preferred over method Al because it is 17 simpler to perform. Is 19 Method A3 Preparation of 100 ml of solUtion containing 90 20 mX Ca. 21 22 This is an adaptation of r.erhod A2. Dissolve 6 grams of 23 the NaCPP commercial phosphopeptide in 20 ml deionised 24 water. Additions are made in the following order except 25 for the two phosphate sol*:ions, which are premixed in SUBSTITUTE SHEET (RULE 36) WO 01/44106 PCT/CB00/04827 24 I the right ratio so that only a single addition is needed. 2 Each addition is made slowly by micropipette with 3 stirring over a period of about 30 min. 4 5 Table 5 Em le additions to form the nanoclumer solution Stock Soluioi Addition Cumulative Volume pH (ml) (mW) H4 20 24 NaN- 3 I 25 NaCl 5 30 NaH 2

PO

4 12 42 NaPO.s 13 55 CaC12 45 100 6.76 6 7 Method A4 Preparation of 4.5 ml of solution containing 8 approw-inately l-M Ca. 9 - 10 This was achieved by ultrafiltration of 50 ml of the II solution prepared by method A3 through a Diaflo 12 ultrafiltration membrane type XM50 in a 10 ml stirred 13 cell (Amicon Ltd, Stonehouse, Glos.) at a pressure of 15 14 pounds per square inch until the final volume was 15 approximately 4.5 ml. The retentate retains the 16 nanocluster particles while allowing the uncomplexed 17 peptides and salts to permeate the membrane. 18 19 Method AS Preparation of very high calcium concentration 20 solutions from freeze dried nanoclusters. 21 ~ 22 'eeze dry 10ml of the solution prepared by method A3 and 23 reconstitute in a suitable volume of water to achieve the 24 desired concentration, for example 0.9ml to give an 25 approximately 1-M solution. By isolating freeze-dried SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCT/GBO0/04827 25 1 nanoclusters and reconstituting them in this way, very 2 high concentrations of calcium can be obtained. 3 4 Methods A4 and A5 are the best method for preparing * 5 concentrations above about 200mM calcium. Method A5 is. 6 expected to be cheapest and to have the potential for the 7 highest calcium concentrations. a 9 Method A6 Peparation of a solution of esential trace 10 elements 12 The formulation contains recommended adult allowances of 13 Ca, Mg, Fe, Cn, Mn, I and Zn in a volume of 300 ml. 14 15 To prepare 100 ml of the solution, dissolve 6 g of the 16 NaCPP commercial phosphopeptide mixture in distilled 17 water to a final volume of 52.2 ml. Add stock solutions 18 in the following sequence, with stirring. 19 Salt Concentration Volume Final (mM) (ml) Concentration !NaH 2

PO

4

.H

2 0 200 10 60mM Phosphate Na3 P04 200 20 60mM Phosphate FeC1 3 .6R 2 0 100 1.0 1.0 mM Fe ZnCl 2 100 0.6 0.6 mM Zn CuSO 4 .5H20 10 0.8 80pM Cu MnSO 4

.H

2 0 10 2.0 200pM Mn KI 1 0.4 4pMI MgCl 2 .6H 2 0 1000 4.0 40mM Mg CaC1 2 . 2M 2 0 1000 9.0 90mM Ca SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCr/CB00/04827 26 2 The solution becomes turbid after the addition of the 3 magnesium and calcium solutions but gradually clears on 4 leaving for 2 days, with stirring. S 6 Freeze drying produced 7.59 g of powder. 7 a The freeze dried, powder redisolves readily in water to 9 form a nearly clear solution with a pH of 7.1. One gram 10 of the freeze dried powder containing 0.74 mg Fe, which 11 can therefore be used in a readily soluble form. 12 Properties of the sample 13 A calcium phosphate nanocluster solution prepared by a 14 method similar to A4 was analysed for phosphopeptides in IS the retentatie and in the ultrafiltrate by reversed phase 16 high pressure liquid chromatography following a 17 modification of the procedure of EllegArd et al. (1999). 18 19 Peptides were separated on a C18 reversed phase Apex 20 Wide-pore column (ex Jones Chromatography Ltd.) Column 21 dimensions were 25cm x 4.4mm internal diameter. An 22 acetonitrile gradient of 5 to 35% over 30 min was used at 23 a flow rate or lml/min and a temperature of 46 degrees C. 24 Trifluoroacetic acid (0.1% v/v) was present in the 25 eluting buffers. Detection was at 214nn. 26 27 Figure 1 shows the resulting chromatograms. Figure la 28. shows the chromatograms for the retentate and 29 ultrafiltrate. Figure lb shows an optimised subtraction 30 of the ultrafiltrate from the retentate to reveal the 31 profile of the fractions bounds to the nanoclusters. 32 SUBSTITUTE SHEET (RULE 26) WO 01/44106 PCr/GB00/04827 27 I The ultrafiltrate chromatogram is closely comparable to 2 that of EllegArd et al. (1999) and shows a wide range of 3 different phosphopeptides. The chromatogram of the 4 retentate is somewhat different and shows the s differential binding of phosphopeptides to the 6 nanocluster particles since only the latter are fully 7 retained during ultrafiltration. Thus it may reasonably 8 be concluded that the nanocluster particles made with 9 the NaCPP are stabilised by a broad mixture of 10 phosphopeptides. 11 12 The formation of nanoclusrcers, nanocluster-like particles 13 or larger particles composed of assemblies of 14 nanoclusters has been established by small angle X-ray or 05 neutron scattering experiments, as described by Holt et 16 al. (1996, 1998). 17 IS Table 6 Confirmation of nanocluster formation by small angle X-ray or neutron scattering Sample Conclusion Whole casein Milky solution confining particles of the size and substructure of native casein micelles. Mixtures of fl- and c-caseins Turbid or milkv solutions depending on the ratio of c- to p-cascins. Size was inversely proportional to the ratio from a minimum radius of gyration of I I nm. At small ratios a substructure is seen closely similar to that of native casein micelles p-casein 5P Uncontrolled precipiation of the f-casein aggregates showing the need for an additional dispersing agem. A commercial phosphopeptide mixture (NaCPP) Clear, stable solutions of nanocluster-like particles supplied by MD foods. could be formed but the panicles were polydisperse with a size range extending from several nanometers to several tens of nanometers SUBSTITUTE SHEET (RULE 26) WOO01/44106 PCriGB00104827 A phouphoprptide mixture derived from a digestion Clear. stable solutions ornanocluster-ike particles of whole casein with protease XIV from could be formed but thec particles were polydisperse Streptm we griseus and papain type IV containing with a size range extending from several nanomters NI-.acsin 2P (A4651), oti -casein 4P 06~1-70). 03- to several tens of nameter main4PQfM 1-2 1), a,,-casin 3P (13-12), %-cascin 4P (f4MI6) aca-cscin zP (n 26-133). A~casein phosphopeptide 4P((-25) Moniodisperse nanoclinters formed (Holt et at. 1996, 1998) IP-cascin phosphopeptide 4P Mt-25) Monodisperse nanocluster formed IA-casein phosphopepuide 5P (fl-42) Monodisperse nanociusws formed (Holt ca a/.. 1996, Cyanogen bromide cleavage fragment asi-casein 2P Peptidc aggregation during the preparation of the (1fI-54) sml Cyanogcn bromide cleavage fragment as I-casin 6P Pepride a;&repaion during the preparation of the ((61-23).sample Tiyptic phosphometide asi-casein I P Vfl04-1 19) Prectpitation of calcium phosphate Trypuc phosphopeptide ctli-casein 2P (M4-59) Precipitation of calcium phosphate Tryptic phosphopeptide tz,-casein 5P (539-79) Clea& solution of stable nanoclusters in the normal size range typtic phosphopeptide ot, 1 -casein IP (f43-79) Clear solution of stable nanoclusters in the normal Size range 1 2 Further modifications and alterations may be made within 3 the scope of the invention herein described. SUBSTITUTE SHEET (RULE 26)

Claims (50)

1. Thermodynamically stable calcium phosphate nanoclusters formed from a nanocluster forming solution wherein the calcium phosphate 5 nanoclusters comprise calcium, phosphate and phosphopeptide and/or phosphoprotein molecules, with a ratio of 10:4.9:3.0, for Ca:P:Po where Pi and Po are inorganic and organic phosphorus respectively. 10

2. Thermodynamically stable calcium phosphate nanoclusters as described in claim wherein the nanoclusters have an approximate empirical formula in the range: [Ca(HPO2- )04_1 0(PO- )-,. 3 (H 2 O), LCa,-SerP, - peptides where (y>3) and the sum of- charges of the ions within both square 15 brackets is approximately zero, and where Ca 2 - 5 SerPy-peptide is the calcium salt of a multiply phosphorylated phosphopeptide molecule.

3. Thermodynamically stable calcium phosphate nanoclusters as 20 described in claims 1 or 2, wherein the nanoclusters contain beta casein phosphopeptide residues 1 to 25, and have the empirical formula: tCa 4 7 (HPO 4 ~), (POt )4,,(H 2 0) 4 .,1 [Ca 7 SerP 4 - Casein| where SerP 4 -Casein is the phosphopeptide. 25

4. Thermodynamically stable calcium phosphate nanoclusters as claimed in claims 1 to 3, wherein the particles are 30 thermodynamically stable in the pH range 6.0 to 7.2 and have a solubility product, Ks given by: K, =8.671(±0.600).10 M 7 " or K, = {Ca2+ 1IOHPO ~ "{P.4j}J467 5 where the curly brackets denote activity.

5. Thermodynamically stable calcium phosphate nanoclusters as described in claims 2 to 4, wherein the nanoclusters comprise 10 to 200 empirical formula units. 10

6. Thermodynamically stable calcium phosphate nanoclusters as described in claims 1 to 5, wherein a plurality of different types of phosphopeptide molecules may be selected from phosphopeptide molecules derived from an enzymatic digest of whole casein 15 preparation.

7. Thermodynamically stable calcium phosphate nanoclusters as claimed in claims 1 to 6 wherein the phosphopeptide molecule has a centre of phosphorylation. 20

8. Thermodynamically stable calcium phosphate nanoclusters as claimed in claims 1 to 6, wherein the phosphopeptide molecule contains two centres of phosphorylation. 25

9. Thermodynamically stable calcium phosphate nanoclusters as claimed in claims 1 to 6, wherein the phosphopeptide molecule contains three or more centres of phosphorylation. 31

10. Thermodynamically stable calcium phosphate nanoclusters as claimed in claims 7 to 9, wherein the centre or centres of phosphorylation have at least three phosphorylated residues. 5

11. Thermodynamically stable calcium phosphate nanoclusters as claimed in claims 7 to 10, wherein the centre or centres of phosphorylation have at least three phosphorylated residues in a series of six consecutive residues in the primary structure of the 10 phosphopeptide molecule.

12. A calcium containing food or beverage comprising thermodynamically stable calcium phosphate nanoclusters as claimed in any preceeding claim. 15

13. A pharmaceutical composition for the inhibition or prevention of tooth deminerallsation, comprising thermodynamically stable calcium phosphate nanoclusters as claimed in any one of claims 1 to 11. 20

14. A pharmaceutical composition for the treatment or prevention of pathological calcification, the composition comprising thermodynamically stable calcium phosphopeptide nanoclusters as claimed in any one of claims 1 to 11, and a pharmaceutically 25 acceptable carrier.

15. A pharmaceutical composition for the treatment or prevention of pathological calcification as claimed in claim 14, wherein the pharmaceutically acceptable carrier comprises an ion permeable 30 matrix. 32

16. A pharmaceutical composition for the treatment or prevention of pathological calcification as claimed In claim 15, where the ion permeable matrix is a gel. 5

17. A pharmaceutical composition for the treatment or prevention of pathological calcification as claimed In claim 16, wherein the ion permeable matrix is a casein based gel. 10

18. A pharmaceutical composition for a soluble mixture of essential central trace elements comprising thermodynamically stable calcium phosphate nanoclusters as claimed in any one of claims 1 to 11, and essential trace elements. 15

19. A pharmaceutical composition for a soluble mixture of essential trace elements as claimed in claim 18, wherein the essential trace elements may be one or more from the list of calcium, iron, manganese, iodine and zinc.

20 20. Calcium phosphate nanoclusters as claimed in claims 1 to 11, wherein a dispersing agent is also included.

21. Calcium phosphate nanoclusters as claimed in ctaims 1 to 11, wherein a preservative is also added. 25

22. Calcium phosphate nanoclusters as claimed.in claims 1 to 11, wherein the nanocluster solution is sterilised.

23. Calcium phosphate nanoclusters as claimed in claims 1 to 11, 30 wherein the nanocluster solution is dried. 33

24. Calcium phosphate nanoclusters as claimed in claim 23, wherein the nanocluster solution is freeze dried. 5

25. A method of making thermodynamically stable calcium phosphate nanoclusters comprising the preparation of a calcium phosphate nanocluster forming solution, wherein the calcium phosphate nanocluster forming solution is prepared by simple mixing of calcium ions, phosphate ions and phosphopeptide and/or 10 phosphoprotein molecules with a concentration ratio of [Ca]/[Po] s 3.1, where Ca is calcium and PO is organic phosphorus, and wherein the pH of the calcium phosphate nanocluster forming solution is adjusted from an initial pH to a final pH by the simple mixing of the calcium phosphate nanocluster forming solution, 15 wherein said simple mixing does not require urease to be present in the calcium phosphate nanocluster forming solution.

26. A method of making thermodynamically stable calcium phosphate nanoclusters as claimed in claim 25, wherein the phosphopeptide 20 or phosphoprotein molecules have at least 3 phosphorylated residues.

27. A method of making thermodynamically stable cajcium phosphate nanoclusters as claimed In claims 25 or 26, wherein the 25 phosphopeptide or phosphoprotein molecules contain no hydrophobic regions.

28. A method of making thermodynamically stable calcium phosphate nanoclusters as claimed in claims 25 to 27, wherein the nanocluster 34 forming solution comprises a mixture of phosphoprotein and/or a mixture of phosphopeptide molecules.

29. A method of making thermodynamically stable calcium phosphate 5 nanoclusters as claimed In claim 28, wherein the mixture of phosphopeptide molecules is an enzymatic digest of a crude casein preparation.

30. A method of making thermodynamically stable calcium phosphate 10 nanoclusters as claimed in claims 25 to 29 any preceding claim, wherein the thermodynamic activities of calcium phosphate ions in the solution formed are large enough to exceed the following solubility product type relationship, such as found in milk: K, {Ca2+XPO4}Of{HPO' 7 ;1.6x -0-7 K, 10- 6 M' 9 or K 8 ={Ca2+O{HPO4- f-{PO-Y-6 15 or K, = 8.671(± 0.600)x 10~9 M 1767 where the curly brackets denote activity.

31. A method of making thermodynamically stable calcium phosphate 20 nanoclusters as claimed in claims 25 to 30, wherein the nanocluster forming solution can be described by: [Ca, ]I[PP] 3;[Pi,]= (0.8750. 125)x [PP)- 1.67 ± 0.25 where [PP] is the concentration of the phosphopeptide in grams per litre, (Pi] is the total millimolar concentration of inorganic phosphate 25 and [Cad is the total millimolar calcium concentration. 35

32. A method of making thermodynamically stable calcium phosphate nanoclusters as claimed in claims 25 to 31, wherein the nanocluster forming solution does not contain significant (greater than 0.1 mM) 5 amounts of further calcium chelating agents.

33. Thermodynamically stable calcium phosphate nanoclusters obtained by the method as described in claims 25 to 32. 10

34. Thermodynamically stable calcium phosphate nanoclusters as claimed in claim 33 wherein the phosphopeptide molecule has a centre of phosphorylation.

35. Thermodynamically stable calcium phosphate nanoclusters as 15 claimed in claim 33, wherein the phosphopeptide molecule contains two centres of phosphorylation.

36. Thermodynamically stable calcium phosphate nanoclusters as claimed in claim 33, wherein the phosphopeptide molecule contains 20 three or more centres of phosphorylation.

37. Thermodynamically stable calcium phosphate nanoclusters as claimed in claims 34 to 36, wherein the centre or.centres of phosphorylation have at least three phosphorylated residues in a 25 series of six consecutive residues in the primary structure of the phosphopeptide molecule.

38. A calcium containing food or beverage comprising thermodynamically stable calcium phosphate nanoclusters as 30 claimed in any one of claims 33 to 37. 36

39. A pharmaceutical composition for the inhibition or prevention of tooth demineralisation, comprising thermodynamically stable calcium phosphate nanoclusters as claimed in any one of claims 33 5 to 37.

40. A pharmaceutical composition for the treatment or prevention of pathological calcification, the composition comprising thermodynamically stable calcium phosphopeptide nanoclusters as 10 claimed in any one of claims 33 to 37, and a pharmaceutically acceptable carrier.

41. A pharmaceutical composition for the treatment or prevention of pathological calcification as claimed in claim 40, wherein the 15 pharmaceutically acceptable carrier comprises an ion permeable matrix.

42. A pharmaceutical composition for the treatment or prevention of pathological calcification as claimed in claim 41, where the ion 20 permeable matrix is a gel.

43. A pharmaceutical composition for the treatment or prevention of pathological calcification as claimed in claim 42,_wherein the ion permeable matrix is a casein based gel. 25

44. A pharmaceutical composition for a soluble mixture of essential central trace elements comprising thermodynamically stable calcium phosphate nanoclusters as claimed in any one of claims 33 to 37, and essential trace elements. 30 37

45. A pharmaceutical composition for a soluble mixture of essential trace elements as claimed in claim 44, wherein the essential trace elements may be one or more from the list of calcium, iron, manganese, iodine and zinc. 5

46. Calcium phosphate nanoclusters as claimed in claims 33 to 37, wherein a dispersing agent is also included.

47. Calcium phosphate nanoclusters as claimed in claims 33 to 37, 10 wherein a preservative is also added.

48. Calcium phosphate nanoclusters as claimed in claims 33 to 37, wherein the nanocluster solution is sterilised. 15

49. Calcium phosphate nanoclusters as claimed in claims 33 to 37, wherein the nanocluster solution is dried.

50. Calcium phosphate nanoclusters as claimed in claim 49, wherein the nanocluster solution is freeze dried. 20