CARIES INHIBITION

Abstract: Compositions for the inhibition of carries and gingivitis, containing a protein or polypeptide particularly phosphoproteins or polyphosphopeptides including those containing the amino acid sequence (X-Y-Z)<un>u where X and Z are a phosphoserine, phosphothreonine, phosphotyrosine, glutamate or aspartate, Y is any amino acid and n is 1 or more. Particular examples of suitable active ingredients are sodium caseinate, calcium caseinate and phosvitin. Suitable proteins or polypeptides were tested via the dissolution rate of hydroxyapatite as measured by the rate of calcium and phosphate released from a hydroxyapatite column.

Description: 'CARIES ' INHIBITION' - 1 - This invention relates to caries iπhibiton. The invention also relates to g-ingivitis inhibition. The present invention provides an orally ingestible composition containing a caries and gingivitis inhibiting amount of a protein or a polypeptide or a salt thereof. Preferably, the protein or polypeptide is a phosphoproteiπ or a polyphosphopepti de . Preferably, the protein or polypeptide is an acidic phosphoprotein or polypeptide. Preferably, the protein or polypeptide contains the amino acid sequence (X-Y-Z) where X and 2 are a phos- phoseriπe, phosphothreoniπe, phosphotyrosine, glutamate or asparate and Y is any amino acid. Preferably, the protein or polypeptide contains a plurality of units each having the amino acid sequence (X-Y-Z) where X, Y and Z are as stated in above- Preferably, the protein or polypeptide contains a group of formula (X-Y-Z)n where X, Y and Z are as stated in cl aim 4 and n s 1 or more . Preferably, n is 3 or more. Preferably, X and Z are phosphoseri ne . Preferably, the protein or polypeptide is a polyphosphoseriπe. Preferably, the phosphate groups of the polyphos- phoserine are spaced at about 6.88 Angstrom Units. Preferably, the protein is a casein. Preferably, the protei Alternatively, the pro Preferably, the protein or polypeptide is in solution Preferably, the protein or polypeptide is one exhibit ing a reduction in calcium dissolution rate of at least 45 nmo /πππ under the test conditions defined herein. 1 Preferably, the protein or polypeptide is one exhibiting a reduction in calcium dissolution rate of at least 80 nraol/min under the test conditions defined herein. Preferably, the protein or polypeptide is one • exhibiting a reduction in calcium dissolution rate of at $ least 90 πmσl/ππn under the test conditions defined herein. Preferably, the protein or polypeptide is one exhibit iπg~a~.reduct oπ .in calcium dissolution rate of at least 95 nmo /rain under the test conditions defined herein. Preferably, the protein or polypeptide is present as 5 up to 10% by weight. Preferably, the protein or pα.l peptide is present as up to 5% by weight. Preferably, the protein or polypeptide is present as up- to 2% by we ght. S* Preferably, the composition additionally contains urea. The composition of this invention may be in the form of a foodstuff, confectionery, dentifrice, tablet or comprise a pharmacolog cally acceptable vehicle, solution of suspensio 25 far topical application to the teeth or a mouthwash. Other modes of administering -the protein or polypeptide would be acceptabl e- f pharmacologically acceptable. Of particular interest as compositions are chewing gum, breakfast foods, ice-cream and other frozen confectioner •30 confectionery, sweets and cakes as these are all known as caries problem foods. Also of particular interest are dentifrices, mouth- washes and preparations for topical application to teeth, α - casein and ' other caseins are obtainable from 35 milk, phosvitin is obtainable from egg yolks and other suitab phosphoproteins include those which are obtainable from cereals, nuts and vegetables particularly in bran husks or sheaths. In particular, rice, wheat, oat, barley and rye brans are a source of such phosphoproteins. Polypeptides of interest include polyphosphoserine , 5: p-olygl utamate and polyaspartate. The present invention also provides a process of iTThi biting dental caries and/or tooth erosion and/or gingiviti comprising applying to the teeth with a carrier a cariostatic and/or antigingi vi tis agent selected from a protein or a poly- 10. peptide or a salt thereof. The present invention also provides a first test procedure for selecting among the proteins or polypeptides that may be used those that are most effective. TEST 1 5 The purpose of this test is to determine hydroxyapatit dissolution and in this respect since tooth enamel is largely composed of hydroxyapatite it is believed that useful compar¬ isons can be made. Double distilled, deionised water (greater than 10 0 MΩ/c ) was used throughout. Analytical reagent grade chemicals were obtained from Ajax Chemicals, Australia. * Hydroxyapati e-spheroi dal was purchased from BDH, and phosviti from Sigma Chemical Co., Missouri, U.S.A. The mil proteins were fractionated by the method of Zittle and Custer (1), 5 and their purity assessed by polyacryl ami de gel electro- phoresis using a modification of the method of Groves and Kiddy (2).. Methods Hydroxyapat te Dissolution Rate Assay 0 ' A chro atography column (pharmacia K9/15) containing 1 g. of hydroxyapatite beads was used for the demiπeral sation assay. Tris 5 M, pH 8.3 containing 50 mM NaCl and 20 mg/1 πeomyciπ was used as the column influent buffer at 20°C and at a rate of 1.000+0.003 ml./min. A protein solution (10 rag. 5 10 ml. of influent buffer) was applied to the column and 1 ml. fractions were collected before and after protein application and assayed for total calcium, phosphate and protein. From these values a rate of dissolution (nmol calciuπ. or phosphat per min) for each \_ ml. fraction was obtained. Calcium, Phosphate and Protein Assays Inorganic phosphate was measured by the method of Itaya and Ui ( 3) and protein by the method of bradford (4). The determination of calcium was fay atomic absorption spectr photoraetry using 1% lanthanum chloride to prevent phosphate suppression . Results The proteins used for the study are listed in Table they are all acidic proteins and included four phosphoprotein and three πoπ-phosphory ated proteins from the whey fraction of bovine milk. The * effect of the individual proteins on hydroxyapati e dissolution rate is shown in Table 2. Table 1. Properties of various phosphoryl ate and noπ-phosphory ated acidic proteins. Protein Molecular Phosphoseriπe Isoelectric Carbohydra Weight Content α Point Conten Phosvitin 35,500 110 1.5 + α s -casein 23,513 8 -.1 - 0-casein 24,020 5 4.5 - κ-casein 19,037 1 3.7 + α-lactalbumiπ 14,174 - 5.1 - 0-lactaglobul in 18,362 - 5.3 - Bovine serum 56,210 - 4.7 - albumin a. Residues per mol protein Table 2. Effect of phosphorylated and non-phosphorylated proteins on hydroxyapatite dissolution rate. Protein Reduction in Re-duction in Amount of protein calcium phosphate bound ' dissolution dissolution rate a rate (nmol/ in) (nmol/min) (mg) Phosvitin 93. l± 5.4' 63.8± 9.4 1.87+0.62 α s -casein 100. l± 4.1 1 63.5± 3.3 5.58±0.03 0-casein 94.8±11.7 I 64.0+19.3 7.45±0.37 - -casein 56.3± 8.9 33.7± 6.8 4.17+0.26 α-lactalbumin 2.7± 1.7 2.9± 0.6 0.48=0.17 β-lactogl obul in 17. l± 1.7 12.5± 1.2 1.8020.71 Bovine serum 31.6± 4.5 20.5± 3.3 2.09+0.05 albumin a. means±SD, n = 3 b. not significantly different P>0.5 In a trial of the above test the dissolution rate of hydroxyapatite as measured by the rate of calcium and phosphate released from the hydroxyapatite column was constan over a two hour period calcium 353.6±3.9 nmol/min, phosphate 225.4+6.8 nmol/min. The dissolution rates obtained using different hydroxapati te columns, showed greater variation, calcium 354.2+23.8 nmol/min, phosphate 229.6±30.8 nmol/min, n = 11. This iπtercolumn variation in dissolution rate could be attributable to different column packing resulting in a different HA surface area exposed. The effect of phosvitin on the dissolution rate of hydroxyapatite is shown in Figure 1. The protein caused an initial increase in the dissolution rate of phosphate which then decreased and approached a new steady state level; 63.8 nmol/min less than the rate prior to phosvitin application. The protein caused an immediate and marked drop in the calcium dissolution rate which then increased and approached a steady- state level 93.1 nmol/min less than that prior to phosvitin application. The amount of protein that passed through the column was measured and from this the amount retained was calculated 1.87 mg. The dissolution rate returned to the original v.alue after phosvitin was eluted from the column with 10 ml of eluent buffer containing 1.5 M phosphate, followed by buffer not containing phosphate. The trace for α_-casein was ery similar to that of phosvitin except that the immediate drop in calcium dissolution rate was not as marked Fig. 2. The differenc in the steady-state rates of calcium and phosphate release 0 before and after α -casein application were very similar to those in Figure 1 (calcium, 100.1 nmol/min. phosphate 63.5 nmol/min). The results * obtained forø-casein (Fig. 3) were characteristic of all the other proteins tested, except 5 for the final steady-state rates of calcium and phosphate released. All proteins {with the exception of phosvitin and α-casein) caused an initial increase in the dissolutio rate of both calcium and phosphate which then decreased as the proteins passed out of the column. The mean 0 differences between the steady-state dissolution rates before and after protein application, together with the amount of protein bound, ' for all proteins tested is presented in Table 2 above. The results show that the four phosphoproteins gave a marked reduction in the 25 steady-state dissolution rate of HA with phosvitin, α s -casein and B-casein all giving the same reduction in calcium and phosphate dossolution. The results show that all the acidic proteins caused a. reduction in the steady-state dissolution rate ■30 of hydroxyapatite. However, the greatest reduction was given by the four phosphoproteins; phosvitin, α -casein, β-casein and to a lesser extent K-casein. SU?.- From the above and from other data which suggests that adjacent phosphate groups of polyphosphoser ne compounds have a spacing of about 6.88 Angstrom units when in a beta-pleated sheet configuration and that calcium atoms in a hydroxyapatite surface along the c-axis will also be spaced at about 6.88 Angstrom units, we speculate that a phosphate group-calcium atom bond materially reduces hydroxyapatite dissolution. References: 1. Zittle, C.A., Custer, J.H.: Purification and some properties of α s -casein, <3. Dairy Sci 46: 1183-1189, 1963 2. Groves, M.L., Kiddy, C.A.: Polymorphism of γ-caseiπ in cow's milk. Arch. Bioche . Biophys 126: 188-193, 1968. 3. Itaya, K., Ui , M.: A new icromethod for the colorimetric determination of inorganic phosphate. Clin, Chim, Acta 14: 361-366, 1966. 4-. Bradford, MM.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254, 1976. TEST 2 For the purpose of in vivo testing, the following test p-rocedure for determining the effect of casein and whey protein on caries incidence in the Sprague-Dawl ey rat. Materials and Methods. Forty-five, weanling, male Sprague-Dawl ey rats, 18 days old, bred from animals fed a fluoride free diet were used. The rats were marked for indentifi cation and then randomly distributed with respect to diet. They were housed in raised-bottom stain ess-steel cages, one group of 15 per cage and fed a powdered cariocenic diet with either deionised water (control), a 1% casein solution or a 2% whey prote n solut on ad libitum. The * cariogenic diet was a modified MIT - 200 diet shown in Table 3. - - -----.-5- ,—* Table 3 - Composition of modified MIT - 200 cariogenic di Component % t Sucrose 67 Wheyproteiπ 20 Concentrate Salt mixture 3 Cottonseed oil 3 Cellulose a 6 Vitamin mixture a * 1 a.. Calcium and phosphate not detectable, fluoride conten of complete diet was less than 0.2 g per g dry weight, b. Vitamin and Salt mixture described in detail elsewher The animals were weighed daily and the amounts of powdered diet and liquid consumed over a 24 h period by each group was measured. After 42 days on the diet, the animals were killed by cervical dislocation and treated as described below. Caries evaluation. Fissure caries was assessed using the method of Konig, Marthaler and Muhlemann ( 1958) ( 5 ) . The mandible was removed from each rat and placed in f or o -sal ine. The jaws were the sectioned and stained by the method of Konig et al (1958)(5), as described by Green and Hartles (1966)(6) to provide series of 100 μm thick longitudinal mesio-distal sections of the molar teeth. Only the main fissures of the first and second molar teeth were assesse for caries. Resul s Diet Consumption The relative consumption of solid and liquid diet fay the three groups of rats was tested by an analysis of variance (by diet). This showed that the quaπitites of both solid and liquid consumed by each group were. not significantly different (P>0.75). - - 9 - Caries Analysis. The caries data shown in Table 4 were analysed in an analysis of variance table by. diet. Table 4 > Caries Experience Data Animals Number of carious fissures Control Group 7.92±2.06 Casein Group 1.87±2.50 Whey Protein Group 4.73+3.85 a. Maximum number possible 10. The animals drinking the 1% casein solution had 76.5% less carious fissures than the control animals (p<0.0 1), and the animals consuming the 2% whey protein solution had 40.3% less carious fissures than the control group (p<0.01). The correlation of caries incidence with the final weight of the rat was .tested for the three group No correlation attained significance (p>0.1). Similarly, the initial and final weights showed no correlation, nor were weight gain and caries incidence correlated . Cone! usi on Acidic proteins in the drinking water substantiall reduced caries incidence of male Sprague-Dawl ey rats, however the phosphorylated protein (casein) caused a greater reduction than the noπ-phosphoryl ated whey protein References : (5) Konig K.G., Marthal e 'T.M. and Muhlemann H.R. 1958 Methodik der Kurzfristig erzeugten Rattenkari es , Dr. Zahn-Mund-u. K.i ef erheil k . 29, 99-127. (6) Green R.M. and Hartles " R.L. 1966: The effect of differing high carbohydrates diets on dental caries in the albino rat. Br. J. Nutr. 20, 317-323. - 10 - • -- TEST 3 This test was to determine the effect of protein on the. adsorption of the bacterium Streptococcus mutans to hydroxyapat te. Materials and Methods Hydroxyapatite discs were prepared by pressing 150 mg of hydroxyapatite (Bio-Gel HTP, Biorad Laboratories for 5 min under 5 tons of pressure in a KBr press. The discs were hydrated then incubated with either various protein solutions or iraidazole buffer (0.05M pH 7.0, containing 0.025 M NaCl). The adherence of 3 H-labelled S.mutans PKl cells was studied by incubating the pretreate discs with 3 H-thym dine labelled cells (10 9 cells/ml) suspended in the i idazole buffer. The protein solutions used were all 5 mg/ l in imidazole buffer. The proteins and polypeptides studied were t s -casein , β-caseiπ, <-caseiπ, phosvitin, bovine serum albumin, histone III, histone VII , α- actalbumiπ, B-lactoglobulin , poly-1 -lysi and poly-1-glutamate. The caseins were prepared by selective precipitation and the other proteins were purchased from Sigma Chemical Co., Missouri, U.S.A. Results The effect of pretreating hydroxyapatite discs withvariαus protein solutions on the adherence of S.mutans cells is shown in Table 5. Table 5. Effect of protein on S. mutans adherence to hydroxyapatite. Proteins Type: Number of S.mutans cell adsorbed (xlO ) Control - 1.9± .6 a α s ι-casein acidic phosphoprotein 0.5±0.3 g- casein acidic phosphoprotein .4±0.4 K - casein acidic phosphoprotein .δ±ø.l phosvitin acidic phosphoprotein 0.5±0.1 BSA . acidic protein β.6±0.1 histone III basic protein 2.2±0.9 histone VIII basic protein 2.6+0.9 O ?I ' Protei ns Type Number of S.mutans cells adsorbed (xlO 7 ) -1 actal bumi n acidic protein 1.0±0.4 -1 actogl obul n acidic protein 0.9±0.4 poly-lysine basic polypeptide 3.8±1.1 poly-gl utamate acidic polypeptide O.δ±O.l a means±5B,n=8 Conclusion All the acidic proteins and polypeptides (especially the phosphoproteins) caused a reduction in bacterial adherence to hydroxyapatite. However, the basic proteins and polypeptides either had no effect or enhanced bacterial adherence to hydroxy¬ apatite. Having regard to the successful results obtained from using the above tests Applicants have formulated various compositions in accordance with this invention as follows. In general , the compositions contain from 0.5-20% by weight of protein or polypeptide. Example 1. Flour: In a device for mixing dry substances, 1% by weight of powdered sodium caseinate was blended with flour. Example 2. Cereal : A breakfast cereal was sprayed with a solution of calcium caseinate in water. The cereal flakes were then dried to produce a finished product containing 1% calcium caseinate. Example 3. Bread: 2% by weight of calcium caseinate was added to the flour during the mixing of ingredients for the manufacture of bread. Example 4. Cake mix: 1% by weight of calcium caseinate was added to the dry ingredients in the preparation of a -cake mix. Example 5. Confectionery: In the preparation of confectionery 2 % by weight of calcium caseinate was added to the final mixture. Example 5. Biscuit: In the preparation of a biscuit mixture 5% by weight of calcium caseinate was added to the other dry ingredients during mixing. Example 7. Beverage: A beverage was prepared in which 1% ^ x- -- - ■< -. - 12 - Example 8. Tablet: A tablet was made containing 10% by weight of calcium caseinate together with excipients being flavouring matter and binding material. In preparation of a typical dentifrice within the scope of this invention, the requisite salt and sa ts of the selected protein or polypeptide are incorporated into dentifrice compositions in any suitable manner depending on whether a powder, paste or liquid preparation is to be produced. For this purpose appropriate preparations of 10 surface-active agents, binders, flavouring materials and other excipients required to achieve the required form of dentifrice are added. The invention is further illustrated by the following examples: Example 9. Tooth paste: A toothpaste was prepared 15 having the ollowing composition:- Calcium caseinate 5.0% by weight Gum tragacanth 1.0% " Saccharin .1% " Glycerin (B.P.) 20. .0% " 20 Sodium lauryl sulphate 1 .0% " Methyl parahydroxy benzoate 0. .1% " Flavouring and colouring 1 .0% " Dibasic calcium phosphate 35 .0% " Water 36.8% 25 Example 10. Toothpaste: A preparation as set out in example 9 was repeated but with the addition of 2% sodium fluoride in a suitable form. Example 11. Toothpaste: A preparation as set out in example 30 9 was repeated but with the addition of 0.4% stannous fluoride in a suitable form Example 12. Toothpaste: A preparation as set out in example was repeated but with the addition of 0.1% mono sodium fluoro- phosphate in a suitable form. ' ---- Example 13. Tooth powder: The following preparation was made:- Calciu caseinate 5.0% by weight Soluble saccharin 0.1% " Colour agent Trace " Dibasic calcium phosphate 94.1% " Example 14. Tooth powder: A preparation as set out in example 13 was made but with the addition of 1% mono sodiu fl uorophosphate in a suitable form. Example 15. Liquid dentifrice: A preparation was made consisting of:- Sodium algiπate 1.5% by weight Calcium caseinate 5.0% Sodium lauryl sulphate 1.0% Flavouring Trace Col ouri πg Trace Water 92.5% pH adjusted to 7.0 Example 16. ' Liquid dentifrice: As for example 15 but wit 0.5% sodium fluoride added. Example 17. Mouthwash: The following preparation was ma.de:- Sodiu caseinate 2.0% by weight Sodium fluoride 0.5% " Flavouring Trace " " Colouring Trace " " Water 97.5% " In the above, casein was used princiaplly because of economics but in lieu phosvitin or other material might be used. The claims form part of the disclosure of this specification. Modifications and adaptations may be made to the above described without departing from the spirit and scope of this invention which includes every novel feature and combination of features disclosed herein.

Claims: 1. An orally ingestible composition containing a caries and gingivitis inhibiting amount of a protein or a polypeptide or a salt thereof. 2. A composition as claimed in claim 1, wherein the protein or polypeptide is a phosphoprotein or a poly- phosphopeptide. 3. A composition as claimed in claim 1, wherein the protein or polypeptide is an acidic phosphoprotein or polypept de. 4. A composition as claimed in claim 1, wherein the protein or polypeptide contains the amino acid sequence (.X-Y-Z) where X and Σ are an phosphoserine, phosphα- threαπine, phosphotyrosine, glutamate or asparate and Y is any amino acid. 5. A composition as claimed in claim 4, wherein the protein or polypeptide contains a plurality of units each having the a ino acid sequence (X-Y-Z) where X, Y and Z are as stated in claim 4. 6. A composition as claimed in claim * 5, wherein the protein or polypeptide contains a group of formula (X-Y-Z) where X, Y and Z are as stated in claim 4 and n is 1 or more. 7. A composition as claimed in claim 6, wherein n is 3 or more. 8. " A composition as claimed in any one of claims 4-7, wherein X and Z are phosphoserine. 9. A composition as claimed in any one of claims 4-8, wherein the protein or polypeptide is a polyphospho- serine. 10. A composition as in claim 9, wherein the phosphate groups of the polyphosphoserine are spaced at about 6.88 Angstrom Units spacing. 11. A composition as claimed in any preceding claim, wherein the protein is a casein. 12. A composition as claimed in claim 11, wherein the protein is a pha-s-casein . O ?I * 13. A composition as claimed in any one of claims 1-10, wherein the protein is phosvitin. 14. A composition as claimed in any preceding claim, wherein the protein or polypeptide is in solution. 15. A composition as claimed in any preceding claim, wherein the protein or polypeptide is one exhibiting a reduction in calcium dissolution rate of at least 45 nmol/min under the test conditions defined herein. 16. A composition as claimed in any one of claims 1-1 wherein the protein or polypeptide is one exhibiting a reduction in calcium dissolution rate of at least 80 nmol/min under the test conditions defined herein. 17. A composition as claimed in any one of claims 1-14, wherein the protein or polypeptide is one exhibitin a reduction in calcium dissolution rate of at least 90 nmol/min under the test conditions defined herein. 18. A composition as claimed in any one of claims • 1-14, wherein the protein or polypeptide is one exhibiting a reduction in calcium dissolution rate of at least 95 nmol/min under the. test conditions defined herein. 19. A composition as claimed in any preceding claim, wherein the protein or polypeptide is present as up to 10% by weight. 20. A composition as claimed in any preceding claim, wherein the protein or polypeptide is present as up to 5% by weight. 21. • A composition as claimed in any preceding claim, wherein the protein or polypeptide is present as up to 2% by weight. 22. A composition as claimed in any preceding claim, and additionally containing urea. 23. A composition as claimed in any preceding claim, in the form of a dentifrice mouthwash, tablet, lozenge or capsule. 24. A composition as claimed in anyone of claims 1-22 in the form of a foodstuff. 25. A composition as claimed in any one of claims 1-22 in the form of confectionery. 26. An orally ingestible composition substantially as hereinbefore described with reference to any one of the Examples. 27. A process of inhibiting dental caries and/or tooth erosion and/or gingivitis comprising applying to the teeth with a carrier a cariostatic and/or anti- gingivitis agent selected from a protein or a polypeptide ora salt thereof. 28. A process of inhibiting dental .caries and/or tooth erosion and/or gingivitis comprising applying to the teeth a composition in accordance with any one of claims 1-26. 29. A composition as claimed in any preceding claim wherein the protein or polypeptide is. resent as 1% by weight or greater. 30. The articles, things, parts, elements steps, features, methods, processes, compounds and compositions referred to or indicated in the specificaiton and/or claims of the application individually or col ectively, and any and all combinations of any two or more of such.

Inventors: MCDOUGALL WALLACE ARTHUR, REYNOLDS ERIC CHARLES
Applicants: MCDOUGALL WALLACE ARTHUR, REYNOLDS ERIC CHARLES, UNIV MELBOURNE, VICTORIAN DAIRY IND AUTHORITY
Application year: 1982
Main classification: A61K7
IPC codes: A23G3, A23G4, A23G9, A61K8, A61K38, A61P1, A61Q11