FI93171B - Method for the production of therapeutically active phosphopeptides - Google Patents

Description

t 93171

Method for preparing therapeutically active phosphopeptides

The application is divided by application 880629 5

The invention relates to a process for the preparation of a therapeutically useful phosphopeptide which prevents the formation of tooth decay.

Phosphopeptides can be used to prevent tooth decay and gingivitis.

The present invention provides a phosphopeptide or a salt thereof, wherein the phosphopeptide has 5 to 30 amino acids, comprising the sequence

A-B-C-D-E

15 wherein A, B, C, D and E are independently phosphoserun, phosphothreonine, phosphotyrosine, phosphohistidine, glutamate and aspartate.

Preferred phosphopeptides are those in which A, B and C

are independently phosphoserine, phosphothreonine, fos- 20 ....

photyrosine and phosphohistidine and D and E are independently phosphoserine, phosphothreonine, glutamate and aspartate.

Particularly preferred phosphopeptides are those in which A, B and C are phosphoserine and D and E are glutamate.

25. The phosphopeptide is preferably in substantially pure form.

The phosphopeptides of this invention and their salts may be useful in the treatment of (i) dental diseases such as tooth decay, gingivitis and dental diseases, (ii) bone thinning diseases such as bone porosity and bone softening diseases, and (iii) mineral malabsorption. prevention.

Accordingly, the present invention provides a composition comprising a peptide or a salt thereof, in accordance with the present invention, and a physiologically acceptable diluent.

• · 2 931 71

The composition may be in the form of a pharmaceutical composition.

The composition may be administered orally.

A mixture of phosphopeptides and / or salts thereof may be used in the composition. In this case, it is preferred that those containing the sequence A-B-C-D-E predominate.

The phosphopeptide or mixture of phosphopeptides is preferably substantially pure, at least to the extent that it does not contain malodorous impurities.

The following phosphopeptides have been found to be useful in the compositions of this invention:

Tl. Glu-Met-Glu-Ala-Glu-Pse-Ile-Pse-Pse-Pse-Glu-Glu-Ile-Val-Pro-Asn-Pse-Val-Glu-Gln-Lys, T2. Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-15 Val-Glu-Pse-Leu-Pse-Pse-Pse-Glu-Glu-Ser-Ile-Thr-Arg, T3 . Asn-Thr-Met-Glu-His-Val-Pse-Pse-Pse-Glu-Glu-Ser-Ile-Ile-Pse-Gln-Glu-Thr-Tyr-Lys, T4. Asn-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-Pse-Pse-Pse-Glu-Glu-Pse-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys and 20 T5. Glu-Gln-Leu-PSE-Pthi-PSE-Glu-Glu-Asn-Ser-Lys.

The amino acid symbols are as follows: Pse-phosphoserine, Ser-serine, Pth-phosphothreonine, Thr-threonine, Glu-glutamate, Asp-aspartate, Ala-alanine, Asn-Asparagine, Gln-glutamine, Gly-glycine, Arg- arginine, His-histidine, Ile-isoleucine, Leu-leucine, Lys-lysnni, Met-methionine, Pro-proline, Tyr-tyrosine, Väl-valine.

The phosphopeptide can be prepared synthetically by chemical synthesis or by genetic engineering, or it can be extracted from a naturally occurring substance.

For cost reasons, it is now more advantageous to extract the phosphopeptide from casein, and in particular from alpha-s-casein or beta-casein. Fosvitine can also be used as a source of peptide. In addition, phosphoproteins from cereals, nuts and vegetables, especially from bran chickens or husks of rice, wheat, oats, barley or rye, can be used to produce the above peptide. Soybeans and meat contain phosphoprotein: 93171 3 which can be used to obtain the above peptide.

Casein, and in particular alpha-s-casein or beta-casein or their salts, such as sodium caseinate, contain polypeptides that can be cleaved into simpler peptides. Such cleavage can be performed by extraction, such extraction can be chemical or proteolytic.

It is presently preferred to extract casein from any of the following, trypsin, pepsin, chymotrypsin, papain, thermolysin or pronase. Of these, trypsin is preferred.

The extracted casein can be fractionated into peptides, comprising the sequence A-B-C-D-E and other peptides. The presence of said other peptides is not detrimental to efficacy, however, certain of said other peptides have an unpleasant taste and accordingly, if any of said other peptides are to be present, it is preferable to remove those having an unpleasant taste.

20 In general, those of said peptides which have an unpleasant taste appear to be hydrophobic.

The following peptides have been found to have an unpleasant taste: 1. Glu-Val-Leu-Asn 25 2. Asn-Glu-Asn-Leu-Leu 3. Ala-Pro-Phe-Pro-Gln-Val-Phe-Gly 4. Leu- Arg-Phe 5. Phe-Phe-Val-Ala-Pro-Phe-Pro-Gln-Val-Phe-Gly-Lys 6. Leu-Arg-Leu 30 7. Phe-Tyr-Pro-Glu-Leu-Phe, (Glu-glutamate; Val-valine; Leu-leucine; Asn-Asparagine; Ala-alanine; Pro-proline; Phe-phenylalanine; Gln-glutamine; Gly-glycine; Arg-arginine; Lys-lysine; Tyr-tyrosine Preferably, the peptide is one that exhibits a reduction in the rate of hydroxyapatite dissolution of at least 15% under the experimental conditions defined herein.

φ 4,931 71

Preferably, the peptide is one that exhibits a reduction in hydroxyapatite dissolution rate of at least 26% under the experimental conditions defined herein.

Preferably, the peptide is one that exhibits a reduction in the rate of hydroxyapatite dissolution of at least 30% under the experimental conditions defined herein.

Preferably, the peptide is one that exhibits a reduction in the rate of hydroxyapatite dissolution of at least 32% under the experimental conditions defined herein.

Preferably the peptide is present in an amount of 0.01 to 10% by weight

Preferably the peptide is present in an amount of 0.01 to 5% by weight

Preferably, the peptide is present in an amount of 0.01 to 2% by weight.

The composition of this invention may be in an edible form, such as a food or confectionery, dentifrice, tablet, or may comprise a pharmacologically acceptable excipient or suspension for topical application to teeth or gingival tissues or for oral washing. Other forms of peptide administration are acceptable as long as they are physiologically or pharmacologically acceptable.

Compositions of particular interest include chewing gum, breakfast foods, ice cream and other frozen confectionery, pastries, confectionery and cakes, all of which are known as tooth decay agents. A similar consideration 25 applies to other potential tooth decay ί food components.

Of particular interest are also dentifrices, mouthwashes and preparations for topical application to teeth and gingival tissue and suppositories for the treatment of bone diseases and mineral malabsorption.

! Also of interest is the use of the compositions of this invention in the treatment of cavities. With respect to the latter, there appears to be evidence of an increase in minerals in the primary lesions considered to be a pre-hole stage. However, there is also evidence to suggest that the use of the compositions of this invention on existing hole surfaces and tooth surfaces formed by removing damaged material from existing holes or breaking is preferred.

In addition, since the use of the composition of the present invention, which is an aqueous solution, on the surfaces of existing holes or on the surfaces of teeth formed by removing damaged material from existing holes or by fracture is likely to have a long-lasting effect, attempts have been made to be this desired long-term effect.

Accordingly, the present invention also relates to a composition of the present invention adapted to remain in contact with a tooth surface for an extended period of time. The present invention also provides methods and means for maintaining the compositions of this invention in contact with a tooth surface for an extended period of time.

With respect to the latter, the extended period of time should be interpreted in accordance with the desired efficiency and the time required to achieve this sufficient efficiency with value. However, in some cases, this extended time may be as short as one day, but is more preferably a period of weeks or months.

In one model, a hole in a tooth is covered with a composition of the invention and the hole is closed to prevent the composition from escaping. Such sealing can be accomplished by covering or using hole filling compositions.

'll In another model, the composition is formulated so that it is applied to remain in place for an extended period of time. In this case, the composition 35 according to the invention may form part of a dental filling composition.

Accordingly, the present invention also relates to a dental filling composition comprising a phosphopeptide of formula A-B-C-D-E, as defined above and applied thereto, which holds the composition 5 on the tooth surface.

Such a dental filling composition may contain dental fillers known per se, including amalgams and curable polymers.

Of particular interest are dental filler compositions containing calcium. The calcium is desirably in the form of calcium phosphate or hydroxyapatite.

The phosphopeptides used in the invention can be separated in many ways, but the use of a fractionation technique is generally preferred.

Phosphopeptides can be separated by fractionation based on molecular size or specific charges. Due to the unique negative charge density and ability to form divalent ion-20 complexes of the peptides represented by the active sequence A-B-C-D-E, as determined, the preferred method of fractionation is anion-ion exchange chromatography or selective precipitation, or a combination of both.

The following methods describe a form of separation.

Separation method

An example of a phosphopeptide is one prepared by trypsin extraction of horn milk from horned milk. Extraction of all sodium caseinate or fractions (alpha-s or beta) prepared by selective precipitation (Zittle, C.A. and Custer J.H .; J. Dairy Sci 46L 30 1183-181, 1963) is performed using protein: trypsin in a 50: 1 ratio. 20 mM Tris HCl pH 8.0, 2.5 mM NaCl at 37 ° C for one hour. Peptides were fractionated using a Pharmacia FPLC system on a Mono Q HR 5/5 column and eluted with a NaCl gradient; buffer A 20 mM Tris HCl pH 8.0, 2.5 mM NaCl; buffer B 20 mM Tris HCl pH 8.0, 500 mM NaCl, gradient 0-100% buffer B / 30 min; II: 7 93171 flow rate 1 ml / min. Fractions were washed and concentrated using an Amicon Ultrafiltration Cell with a UM05 filter. Peptides were identified using the Water Associates PICO-TAG amino acid analysis system, using phenyl isothio-5 cyanate amino acid derivation. Phosphate was determined by the method of Itaya and Ui (Clin. Chim. Acta. 14: 361-366, 1960). The peptides were sequenced (after phosphate removal by alkaline phosphatase using manual

The Edman method and the resulting PTH amino acids were identified using reverse phase HPLC on a Zorbax ODS column 25 x 0.46 cm (DuPont).

The following phosphopeptides were obtained individually by trypsin extraction of sodium caseinate using the above procedure.

15 Tl. Met-Glu-Glu-Ala-Glu-Ile PSE: PSE PSE PSE-Glu-

Glu-Val-Ile-Pro-Asn-PSE-Val-Glu-Gln-Lys.

T2. Glu-Leu-Glu-Glu-Asn-Leu-Val-Pro-Gly-Glu-Ile-Ile-Val-Glu-Leu-PES PES PES PES-Glu-Glu-Ser-Ile-Thr-Arg.

T3. Asn-Thr-Met-Glu-His-Val-Pse-Pse-Pse-Glu-Glu-20 Ser-Ile-Ile-Pse-Gln-Glu-Thr-Tyr-Lys.

T4. Asn-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-PES PES PES-Glu-Glu-PES-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys.

T5. Glu-Gln-Leu-PSE, PSE Pth-Glu-Glu-Asn-Ser-Lys.

In addition, the following peptides were also obtained: 25 T6. Asp-Ile-Ile-Gly-Glu-PES PES-Thr-Glu-Asp-Gln-Ala

Met-Glu-Asp-Ile-Ile-Lys.

T7. Val-Pro-Gln-Leu-Gln-Ile-Val-Pro-Asn-PSE-Ala-Glu-Glu-Arg.

T8. Thr-Val-Met-Asp-Glu-PES-Thr-Glu-Val-Phe-Thr-Lys. 30 T9. Pth-Leu-Glu-Glu-Lys.

Peptides T1, T6 and T7 were also obtained from alpha-caseinase; (including alphagl's and alfag0's) from TPCK trypsin extraction. Peptide T2 was also obtained from TPCK trypsin extraction of beta-caseinate. Peptides T3, T4, T5, T8 and T9 were also obtained from TPCK trypsin extraction of alfag2 caseinate (comprising alfag2, alfag3, alfag4 and alfagg). The amino acid symbols are 8,931 71 as follows: Pse-phosphoserine, Ser-serine, Pth-phosphothreonine, Thr-threonine, Glu-glutamate, Asp-aspartate, Ala-alanine, Asn-asparagine, Gln-glutamine, Gly-glycine , Arg-arginine, His-histidine, Ile-isoleucine, Leu-5 leucine, Lys-lysine, Met-methionine, Pro-proline, Tyr-tyrosine, Val-valine.

Separation method II

The following method describes one selective precipitation model.

The sodium caseinate solution was extracted with trypsin (50: 1, casein: trypsin) for one hour at 37 ° C while maintaining the pH at 8.0 by the addition of NaOH. HCl (0.1 M) was then added to the solution at room temperature to pH 4.7 and the resulting precipitate was removed. BaCl 2 was added to the solution above sa-15 to 0.25% w / v, followed by an equal volume of absolute ethanol and the resulting precipitate. removed and dried. The precipitate was dissolved in one tenth of the original volume of water (the pH was raised to 20 with NaOH to facilitate dissolution) and the solution was acidified to pH 3.5 with HCl. An equal volume of acetone was added and the precipitate was removed and dried. The precipitate was then redissolved in H 2 O and acidified to pH 2.0 by the addition of HCl. The resulting precipitate was removed and discarded and the pH of the solution above sa-25 was adjusted back to: 3.5 with NaOH and an equal volume of acetone was added. The resulting precipitate was collected, redissolved in water and H 2 SO 4 was added to precipitate BaSO 4, which was discarded. The solution above the precipitate was then dialyzed and lyophilized or spray dried. This method gave a mixture of five phosphopeptides.

The phosphopeptides obtained are as follows: «

Tl. Met-Glu-Glu-Ala-Glu-Ile-PES PES PES PES-Glu-Glu-Ile-Ile-Val-Pro-Asn-PES-Val-Glu-Gln-Lys.

35 T2. Glu-Leu-Glu-Glu-Asn-Leu-Val-Pro-Gly-Glu-Ile-

Val-Glu-Leu-PSE PSE PSE PSE-Glu-Glu-Ser-Ile-Thr-Arg.

il 9 93171 T3. Asn-Thr-Met-Glu-His-Val-PES PES PES-Glu-Glu-Ser-Ile-Ile-PES-Gln-Glu-Thr-Tyr-Lys.

T4. Asn-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-PES PES PES-Glu-Glu-PES-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys.

5 T5. Glu-Gln-Leu-PSE, PSE Pth-Glu-Asn-Ser-Lys.

The ratio of phosphopeptides (T1: T2: T3: T4: T5) in the final preparation depends on the starting material and the hydrolysis conditions. Extraction of sodium caseinate with TPCK-trypsin yields mostly T2 with small amounts of T1, T3, and T4.

However, T2 shows greater instability than other peptides, so that more vigorous extraction, as occurs in some commercial casein extracts, yields a preparation containing mostly T1 with small amounts of T3 and T4.

If alpha-s1-casein is used instead of sodium caseinate in this process, pure T1 is obtained.

When beta-casein is the starting material, pure T2 is obtained.

The most common sequences of active peptides are the pentapeptide Pse-Pse-Pse-Glu-Glu. The distances of the phosphate and carb-20 oxyl groups in the beta conformation of this pentapeptide are shown in Figure 1.

The 6.88 Angström distance of phosphates and carboxyls allows specific attachment to calcium atoms along the c-axis of hydroxyapatite crystals. This Penta-25 peptide sequence occurs in peptides T1 to T4 and occurs modified in peptides T5 to Pse-Pth-Pse-G'lu-Glu, followed by careful replacement of phosphoserine with phosphothreonine.

Careful substitutions in the active sequence would be the replacement of phosphoserine with phosphothreonine and, to a lesser extent, phosphotyrosine or phosphothyridine, although phosphoserine is preferred. Another possible replacement of phosphoserine could be with glutamate or aspartate, but again phosphoserine 35 is preferred. A possible substitute for glutamate is as-partate.

10,931 71

The active peptides may form complexes with calcium phosphate and other divalent metal ion salts. One mole of T1 binds 16 moles of CaHPO4 so that a 10 mg / ml T1 solution at pH 7.0 can dissolve 60 mM CaHPO4, forming a metastable supersaturated solution relative to calcium phosphate compounds. When chloride is the counterion, 1 mole of T1 binds only 5 moles of Ca ++, which binds only to serine phosphates. One mole of T1 bound to 16 moles of CaHPO4 (M.W. 4883) is hereinafter referred to as calcium phosphate

Tl, respectively. An important chemical property of calcium phosphate T1 is that it is more than 2% w / v. the aqueous composition contains a thixotropic gel. T1 to T5 have been shown to be potentially anti-rot agents using the following test methods:

Tests 1

Hydroxyapatite dissolution rate test This test is a modification of the test method already described (Raynolds, E.C., Riley, P.F. and Storey, E. Calcif. Tiss Int 34: s52 -20 s56, 1982). The purpose of this experiment is to determine the effect of peptides on the dissolution of hydroxyapatite and in this regard, since tooth enamel is largely composed of hydroxyapatite, it is believed that useful comparisons can be made.

Twice distilled deionized water was used. (18 mega ohms / cm). Pure analytical reagent chemicals were obtained from Ajax Chemicals, Australia. Spherical hydroxyapatite was purchased from BDH. A chromatography column containing 0.1 g of hydroxyapatite beads was used in 30 mineral removal experiments. Tris 5 mM, pH 8.3, containing 50 mM NaCl was used as a column buffer at 20 ° C and pumped through the column at a rate of 0.1 ml / min. The peptide solution in 0.1 mg / ml buffer was applied to the column and 0.2 ml fractions were collected both before and after the use of peptide-35 and analyzed for total calcium, phosphate and peptide. From these u 93171 values, the dissolution rate (nmol calcium or phosphate / min) was obtained for each 0.2 ml fraction.

All phosphopeptides T1 to T5 reduced the hydroxyapatite solubility rate by about 32%.

Phosphopeptides T6 to T9 were found to be significantly less effective.

Fluoride and phosphopeptide T1 gave a combined decrease in hydroxyapatite solubility (40% decrease). Phosphopeptide T1 caused a 50% higher fluorine retention on the hydroxyapatite column.

This work demonstrates that these phosphopeptides bind to hydroxyapatite and reduce the rate of dissolution of minerals and improve fluoride retention in the crystalline binder. The decrease in solubility of hydroxyapatite was related to the phosphoserine content and the distances between the peptides.

Tests 2

Oral tooth decay test

The anti-tooth decay effect of the phosphopeptide T1 was determined using a modification of the Koulourides and Ostrom oral tooth decay test (Caries Res. 10: 442-482, 1976). The enamel plates were placed in a movable oral device to mimic the space between the teeth. This was done on both sides of the portable device (left and right). The device was worn to allow · * plaque to accumulate in the areas that mimic the space between the teeth. Eight times a day, the device was removed and placed in a 37 ° C solution. The solution contained 2% w / v sucrose, 2% w / v glucose, 140 30 mM KCl, 20 mM NaCl at pH 7.0. Twice a day, the right enamel plates were exposed to a solution containing 1.8% w / v calcium phosphate T1 in 140 mM KCl, 20 mM NaCl at pH 7.0 while the left side was exposed to saline only. At the end of the experiment, the ema-35 plates were removed, cross-sectioned, and subjected to micro-radiography and micro-hardness tests. Microradiography 12,931 71 showed that the plates exposed to the sugar-saline solution (left side) had subsurface, tooth-like-like lesions. However, plates exposed to sugar-saline and peptide T1 solution twice daily showed no tooth-rot-like changes at all. The results were confirmed by micro-hardness analysis. Plaque was also taken from both sides of the instrument and analyzed for calcium phosphate, serine phosphate, and peptide T1 using a competitive quantitative enzyme-linked immunosorbent assay (ELISA) using anti-peptide T1 monoclonal antibody.

This indicated that the plaque on the right side of the device, exposed twice daily to the peptide-T1-15 solution, contained at least 0.4% w / w peptide / wet weight of plaque and had a 2- to 4-fold increase in calcium phosphate content.

This work showed that peptide T1 has associated with plaque and thus increased the calcium and phosphate-20 tip content of the plaque, thus inhibiting the dental decay process. This method of aggregation and accumulation in dental plaque can be used to re-transfer minerals and antibacterial ions into plaque and enamel, e.g., Ca, PO4, FPO4 / Zn, Cu, Sn, Ag, Al, Fe, and La.

25 Tests 3

Oral mineral addition An oral device similar to the previous test method was used, except that the enamel plates were pre-exposed to a mineral depletion solution that caused two subsurface mineral depletion damage. on the plate. The demineralizing solution contained 0.1 M lactate buffer pH 5.0 containing 500 mg / L hydroxyapatite and 1% agar. Subjects used 35 devices for 10 days. Twice a day, the devices were removed and a drop of mineral addition solution was placed on 13 931 71 enamel plates on the right side of the device. The left side of the enamel plates served as a control. After 10 days, the motherboards were removed, cross-sectioned, and subjected to micro-radiography. The amount of mineral deposited back to the subsurface damage sites was determined using a microdensitometer. Mineral supplementing solution containing 1.8% w / v. calcium phosphate T1, pH 7.0, restored 57% of mineral loss compared to 13% in saliva alone.

10 Tests 4

Decreased plaque pH

Subjects abstained from oral hygiene for 3-5 days, followed by a one-minute rinse with 5% sucrose solution. Plaque samples were removed and the pH was measured using the one drop method.

After about five minutes, the pH dropped to about 5.0. However, if subjects were rinsed with a solution containing 1.8% w / v. calcium phosphate T1, pH 7.0, 15 minutes before rinsing with 5% sucrose solution-20a, the pH of the plaque did not fall below 6.7, indicating considerable pH buffering of calcium phosphate T1.

Since the exact mechanism by which phosphopeptides show anti-tooth decay activity is not known, the following speculative theories are presented below, but should not be construed as binding or limiting.

· Phosphopeptides can accumulate in plaque and enamel, buffer plaque acid, prevent mineral depletion from tooth enamel and increase mineral accumulation. The low molecular weight of phosphopeptides may cause penetration and accumulation in the pores of the plaque and enamel. Phosphopeptides, due to the appropriate spacing between serine-phosphate residues, can bind tooth enamel minerals and prevent mineral depletion. Peptides can also transfer calcium and 35 phosphate (with fluorophosphate modification) to plaque and enamel, in a suitable form possibly allowing 14 931 71 spontaneous mineral proliferation. Phosphoserine residues can also buffer plaque acid. The phosphopeptide can also transfer antibacterial metal ions, e.g. Zn, Cu, Sn, Ag, Al, Fe and La, to plaque and in this way has anti-plaque and anti-gingivitis activity. Metal ions are migrated primarily in phosphopeptides, due to phosphoserine residues. Phosphopeptides can bind plaque bacteria and prevent sugar utilization.

The ability of these peptides to form ionic complexes with calcium phosphate can be exploited in the treatment of various rare bone diseases. These peptides can significantly increase the intestinal absorption of calcium, phosphate and iron. Therefore, pharmaceutical excipients (e.g., suppositories) or foods containing calcium phosphate T1 and ferrophosphate T1 can be used to treat osteoporosis / bone softening disease and anemia.

Applicants have formulated various compositions in accordance with this invention in the following. In general, the compositions contain 0.01 to 10% by weight of phosphopeptide.

Example 1

Flour:

In an apparatus for mixing solids, it was mixed with 1% by weight of calcium phosphate T1 flour.

. * Example 2

Cereal Preparation:

The breakfast cereal preparation was sprayed with a solution of calcium phosphate T1 in water. The cereal flakes were then dried to give the final product containing 1% calcium phosphate T1.

Example 3

Bread: 1% by weight of calcium phosphate T1 was added to the flour while mixing the ingredients to make bread.

tl, 15 93171

Example 4

Cake powder: 1% by weight of calcium phosphate T1 was added to the ingredients in the preparation of the cake dough.

5 Example 5

Confectionery:

In the preparation of confectionery, 1% by weight of calcium phosphate T1 was added to the final mixture.

Example 6 10 Biscuit:

In preparing the biscuit / mixture, 1% by weight of calcium phosphate T1 was added to the other dry ingredients during mixing.

Example 7 15 Soft drink:

A soft drink in which 0.1% by weight of calcium phosphate T1 was dissolved was prepared.

Example 8

Tablet: A tablet was prepared containing 10% by weight of calcium phosphate T1 together with excipients which were flavors and binders.

The salt required for the preparation of a conventional dentifrice in accordance with the scope of this invention and the selected salts of the phospho-peptide are combined with the dentifrice compositions in any suitable manner depending on whether a powder, paste or liquid preparation is prepared.

For this purpose, suitable preparations such as surfactants, binders, flavors and other necessary excipients are added to provide the required form of dentifrice.

The invention is further illustrated by the following examples:

Example 9 35 Toothpaste:

A toothpaste was prepared having the following composition: V · 16 93171

Weight-%

Calcium phosphate Tl 5.0 CMC 7MF 1.0

Saccharin 450 0.2 5 Glycerin (B.P.) 25.0

Sodium lauryl sulphate (Empicol 0919) 5.0

Sodium benzoate 0.5

Flavor 9/693090 0.8 10 Calcium phosphate 1.0

Deionized water 39.5

Thixosyl 33J 9.5

Syloid Al-1 12.0

Titanium Dioxide 3328 0.5 15 Example 10

Toothpaste:

The preparation as described in Example 9 was repeated, but 0.2% sodium fluoride in the appropriate form was added.

20 Example 11

Toothpaste:

The preparation as described in Example 9 was repeated, but with the addition of 0.4% tin fluoride in a suitable form.

25 Example 12. Toothpaste:

The preparation as described in Example 9 was repeated, but 0.76% monosodium fluorophosphate was added in the appropriate form.

30 Example 13

Toothpowder:

The following preparation was made: ti: 17 93171 calcium phosphate T1 5.0% by weight soluble saccharin 0.1% by weight dye small amount divalent calcium phosphate 94.1% by weight 5 Example 14

Toothpowder:

A preparation was made as described in Example 13, but 0.76% monosodium fluorophosphate in a suitable form was added.

10 Example 15

Liquid dentifrice:

A preparation was made containing: sodium alginate 1.4% by weight calcium phosphate T1 2.0% by weight 15 sodium lauryl sulfate 1.0% by weight flavor small amount of colorant small amount of water 95.5% by weight

Example 16 20 Liquid dentifrice:

As in Example 15, but 0.5% sodium fluoride was added.

Example 17 Mouthwash: The following preparation was made: calcium phosphate T1 2.0% by weight sodium fluoride 0.5% by weight flavor small amount colorant small amount 30 water 97.5% by weight

Example 18

Carbonated soft drink: 0.1% by weight of calcium phosphopeptide T1 was added to a commercially available carbonated soft drink.

93171 BC

Example 19

Fruit juice: 0.1% by weight of calcium phosphopeptide T1 was added to commercially available fruit juice.

5 Example 20

Solution for topical application to the teeth: calcium phosphate Tl 2%

NaF 0.6 mM

Zn acetate 0.1 mM

10 SrCl2 0.1 mM

(this solution can be formed into a gel by increasing the amount of calcium phosphate T1).

Example 21

Dental filler 15 calcium phosphate Teaspoon 5% w / w calcium phosphate 95% w / w polymerizer small amount

Make a paste with water.

The polymerizing agent used in this example was glutaraldehyde.

Example 22

Dental filler: calcium phosphate Tl 5% w / w calcium phosphate 70% 25 acrylic polymer 25% catalyst for polymer small amount>

Example 23

Topical gel for the treatment of hypersensitive teeth: calcium phosphate Tl 4.0% by weight 30 SrF2 1.0% by weight flavor small amount of water 95%

In the former, calcium phosphate T1 was used for imaging purposes, but any suitable phosphopeptide and / or salt may be used instead.

• · 19 931 71

Modifications and embodiments may be made without departing from the spirit and scope of the present invention, which encompasses all novel features and combinations of features discussed herein.

The claims form part of the presentation of the present invention.

«• ·

Claims (4)

93171 Patented suction A process for the preparation of a therapeutically useful and anti-decay phosphopeptide having the sequence: Glu-Met-Glu-Ala-Glu-Pse-Ile-Pse-Pse-Pse-Glu-Glu-Ile-Val-Pro-Asn-Pse -Val-Glu-Gln-Lys; Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Pse-Leu-Pse-Pse-Pse-Glu-Glu-Ser-Ile-Thr-Arg; Asn-Thr-Met-Glu-His-Val-Pse-Pse-Pse-Glu-Glu-Ser-Ile-Ile-Pse-Gln-Glu-Thr-Tyr-Lys; Asn-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-PES PES PES-Glu-Glu-PES-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys; or Glu-Gln-Leu-Pse-Pth-Pse-Glu-Glu-Asn-Ser-Lys, or a salt thereof, characterized in that an extract of casein, as-casein, β-casein or a salt thereof is formed and the phosphopeptide is separated from the extract by selective aggregation and separation by precipitation or based on the molecular size or charge of the phosphopeptide. Process according to Claim 1, characterized in that the phosphopeptide is prepared in the form of a salt. Process according to Claim 2, characterized in that the phosphopeptide 25 is prepared in the form of the sodium salt. Process according to one of Claims 1 to 3, characterized in that the phosphopeptide is prepared in substantially pure form. tl; 93171