AU599387B2 - Dentifrice compositions comprising hydroxyapatite in which glucanase is immobilized therein - Google Patents

Description

FORM 10 ~f 7 4 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: 1198 documnt contains t anwmdweats mdec tlder s ctioa 49.

and il m et for prinzting.

09 1 00. Name and Address of Applicant: 1P 0 0 0 000 00 0...Address for Service: Dental Kagaku Kabushiki Kaisha Tsukijichuo Bldg.

2-11-10, Tsukiji Chuo-ku, Tokyo

JAPAN

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia 7'

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43 00 0' 0 0 *0 00 a Complete Specification for the invention entitled': _Dentifrice compositions The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 Abstract of the disclosure Disclosed is a dentifrice composition for preventing dental caries, the dentifrice composition containing hydroxyapatite in 'which glucanase is immobilized.

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1 1 j r )69 at a *O 666 Title of the Invention Dentifrice compositions conp 'o \xosioe.

Background of the Invention Field of the Invention This present invention generally relates to a dentifrice composition for preventing dental caries and, more particularly, to a dentifrice composition containing hydroxyapatite in which glucanase and the like are immobilized.

"Glucanase" is a general term for glucan decomposing enzymes.

However, it is understood that the term "glucanase" used herein refers to levanase which decompose levan, dextranase which decompose dextran, and mutanase which decomposes mut an.

Description of the Prior Art It is well-known that the occurrence of dental caries is due to dental plaque which is formed on the surface of teeth by way of polysaccharides produced by a variety of oral bacteria. It is believed that the prevention of dental caries is achieved by the removal of such dental plaque. Levan, mutan and dextran are all polysaccharide produced by oral bacteria, and it has been ascertained that they are a factor in the formation of dental plaque.

Of the enzyme which decompose such polysaccharides, it is well known that dextranase provides a dentifrice composition having an improved effect for prevention of I t rj 6e a *i a SI L

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r 12 The claims defining the invention are as follows: dental caries, since it is capable of dissolving dental plaque due to its dextran-decomposing capability, as disclosed in the specification of Japanese Patent Registration No, 782,154. When the enzyme is incorporated in a dentifrice products, however, it is easily decomposed and deactivated owing to its relative instability. To keep it in a stable state, use of an appropriate stabilizer is required.

Dextranase in also used with a variety of stabilizers.

For instance, it has been proposed to use dextranase O with a terpene hydrocarbon or aliphatic alcohol base perfume (Japanese Patent Registration No. with carvone or S, -mentol in a specific ratio (Japanese Patent Application t 6 Laid-Open No. 56-110609), or with aluminium oxide to be used as a polishing agent (Japanese Patent Application Laid Open No. 56-63915). It may be presumed that use of an enzyme, to say nothing of dextranase, for a dentifrice e on S .O would require the incorporation of a stabilizer as is aQ the case with dextranase. Since a dentifrice is used in a the mouth, it should be safe and impart a refreshing O. feeling. As a matter of course, considerable restrictions S are presumed to be imposed upon the selection of stabilizes.

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For this reason, dentifrice compositions containing mutanase or levanase have not been realized, despite the fact that C| ithey are considered effective in the prevention of dental Scaries.

Summary of the Invention An object of the present invention is to provide C 'a N 0T 2 .4

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a dentifrice composition containing as enzyme, particularly a polysaccharide-decomposing enzyme useful for the prevention of dental-caries, more particularly levanase, dextranase or mutanase, which eliminates problems related to safety and user sensation arising from the use of stabilizers, and which exhibits stable enzymatic activity over an extended period of time without recourse to use any stabilizer. Since enzymes are relatively unstable and soluble, it is well-known that it is better to immobilize them for more efficient use. Generally, immobilized enzymes are found to be stable even in the form of an aqueous solution. While there are a variety of methods for immobilizing enzymes, the classic method relies upon 4 V physical adsorption. As is the case with active carbon, kaolinite, terra abla and the like, hydroxyapatite is used Sas the carrier for immobilizing enzymes by physical C C, adsorption and is known to be suitable for use as a polishing Sagent. It is thus presumed that if levanase, dextranase and mutanase are immobilized with hydroxyapatite, it might o then be possible to prepare an enzyme-containing dentifrice Scomposition which dispenses with any stabilizer. As a result of intensive studies made on such presumption, the present inventors have succeeded in developing a method Si of immobilizing these enzymes with hydroxyapatite. That is, hydroxyapatite used as a polishing agent is added to the solution containing glucanase and protein such as lysozyme, -3- -4albumin, casein and cytochrome C which can be strongly absorbed to hydroxyapatite and which are harmless to human body, then glutaraldehyde solution is added dropwise to those solution under vigorous stirring at below room temperature, and the solution is stirred for several hours to complete the reaction at same temperature after the addition of glutaraldehyde solution. The immobilized hydroxyapatite is obtained by the centrifutation.

Thus according to this invention there is provided a dentifrice composition containing hydroxyapatite having a glucanase or the like immobilized therein.

As the immobilized hydroxyapatites are easily prepared by this process, dentifrice compositions that do not contain a stabilizing agent but glucanase and that have an improved effect upon the prevention of dental caries are prepared by using such hydroxyapatite as the polishing agent according to the conventional dentifrice formulation recipe.

*Table 1 shows the glucanase immobilized hydroxyapatite prepared by the method shown in the specification.

04 /9 of 1 0 z^a *ea aa a ABLE 1 Type Amt. of Apatite No. Type Amt. of Protein Type Amt. of Glucanase GI utaraldehyde 1., 2.

3.

4.

7.

8.

12.

13.

lysozyme O.lg lysozyme 0.lg casein O.1g cytochromie C 0.1g, lysozyme O.lg lysozyme O.Ig 1ysozyme 0.1g lysozyme 0.2g albumin 0.5g lysozyme 0.059 lysozyme 0.05g lysozyme 0.025g lysozyme 0.0125g levanase 0.lg levanase 0.lg levanase 0.lg levanase 0.Ig levanase 0.lg mutanase 0.Ig mutanase 0.lg dextranase 0.2g dextranase 0.05g dextranase 0.2g dextranase 0.2g dextranase 0.025g dextranase 0.0125g dextranase 0.05g hydroxyapatite 2g hydroxyapatite 2g hydroxyapatite 2g hydroxyapatite 2g hydrkxyapati te 2g hydroxyapatite 2g hydroxyapatite 2g hydroxyapatite 5g hydroxyapatite 5g hydroxyapati te 59 hydroxyapatite 5g hydroxyapati te 2.5g hydroxyapatite 2.5g hydroxyapatite 59 1. 12mg 0.56mg 0.56mg 0.56mg /4.48mg 8. 96mg 18.0mg 0 S. Oa~g 0.5-.19 10mg 50mg 50mg 0. 135mg 10.3mg 9.7mg 10.3mg 13.2mg 15.3mg 16.4mg 17.4mg 31.5mg 10. 9mg 9.94mg 15.2mg 8.75mg 12.37mg 0.47g 0.42g 0. 44g 0-499 0.32g 0. 13g 0.02g 0. 0.51g 0. 62g 0.46g 0.06g 0 14. cytochrome C 0.05g 12.15mg 0.46g total banded protein/grams of apatite amount of substrate decomuposition/graffs of protein TMS/91 Ic I I 4b For a better understanding of the invention and to show how the same may be put into effect, reference will now be made, by way of example, to the following working and reference examples.

Example 1: Paste Dentifrice (weight Levanase-immobilized hydroxyapatite Calcium phosphate CMC sodium salt Carrageenan Glycerin 13.2 25.0 0.3 1.2 10.0 ccc M o Oa 4 51 Q aa :19

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TMS/911c Sorbitol 15.0 Sodium lauryl sulfate Perfume 1L.2 Saccharin sodium-salt 01 silica water 30.0 Example 2 :Paste Dentifrice Mutanase-immobilized hydroxyapatite 20.0 ~*Calcium pyrophosphate. 10.0 0MG sodium salt tlac Garrageenan0.

Glycerin 20.0 Sorbitol 10.0 Sodium lauryl sulfate Perfume Saccharin sodium salt 0.1 Silica Potassium chloride Magnesium chloride 0.3 S Sodium phosphate Water 30.0 Example 3: Paste Dentifrice Dextranse-immobilized hydroxyapatite 35.5 0MG sodium salt Garrageenan 0.3 Glycerin Sodium lauryl sulfate Perfume Saccharin sodium salt Silica Sodium chloride Magnesium chloride Potassium chloride Water 35.5 0.1 2.

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20I0 4,~4 4 4 4404 0 4 o 4 4 0440 4 4004 44 4, 4 444 44 44' 444 4 4,4 04 4 4 44 o 4.4 43 4 44*44 44 44 44 4 4 4 44 41 4 t~ Example 4 :Powder Dentifrice Dextranase-immobilized hydroxyapatite Sodium lauryl sulfate Perfume Saccharin sodium salt Sodium chloride Magnesium chlori de Example 5 :Lubricating Dentifrice Dextranznse-immobilized hydroxyapatite Calcium phosphate Sorbitol Sodium laurly sulfate Perfume Sodium chloride Magnesium chloride 90.8 0.2 63.0 10.0 10.0 3.3 0.08 -6 ;a Saccharin Water 0.12 10.0

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Reference Example 1, Preparation of Dextranase-immobilized hydroxyapatite Five hundred (500)mY of potassium phosphate buffer solution having a concentration of 0.05 moles and a PH value of 6.8 was added to a mixture of 50mg of dextranase, of lysozyme, and 5g of hydroxyapatite used as a polishing agent. Five hundred (500)/,0 of a 0.2% aqueous solution of glutaraldehyde was added dropwise under agitation at 4°C to the resulting solution, and stirring was thereafter continued at 4°C for 5hr. The reaction product was collected and was washed three times with 100mfof the aforesaid buffer solution to remove any unreacted matter, Subsequent freeze-drying yielded 5.06g of a powder. About lg of the aforesaid powder was weighed exactly and twenty (20)mR of a potassium phosphate buffer solution having a concentration of 1 moles and a PH value of 6.8 was added to the weighed powder, and stirring was carried out for 3hr, followed by centrifugation. The precipitate was washed with buffer solution and the filtrate were combined to measure the amount of protein contained therein by the lowry method, and the precipitate was washed with pure water to be dried and weighed. It was confirmed that 10.9mg protein was adsorbed to gram of the 44 0 P0 040 4O £1 YY. ~d 7 Ii i dried hydroxyapatite. Measurement of the dextianase activity of such protein by a method to be described later indicated that 0.513g of dextran per gram of the adsorbed protein was decomposed within 2hr.

Preparation of Levanase-immobilized hydroxyapatite Fifty (50)mJ of pure water was added to a mixture of 100mg lysozyme, 100mg levanase and 2g hydroxyapatite, followed by cooling down to 4*C. Two (2)mj of an aqueous solution containing 28mg glutaraldehyde in 300m 9 water was slowly added dropwise under vigorous agitation to the resulting solution, while the temperature of 4 0 C was maintained.

Thereafter, stirring was continued at that temperature for 2hr. Centrifugation yielded a solid which was washed three times with 50mR pure water and then subjected to freeze-drying to obtain 2.05g powder. Analysis effected in the same manner as in indicated that 11.7mg of protein was bonded to gram of hydroxyapatite. Measurement of the levanase activity of the adsorbed protein, effected by the following method, indicated that 0.47g of levan was decomposed per gram of the protein adsorbed to hydroxyapatite.

Preparation of mutanase-immobilized hydroxyapatite The same conditions as in were applied, except that mutanase was used in place of levanase, to obtain immobilized hydroxyapatite. Analysis effected in the same manrer as in indicated that 15.0mg protein was bonded to per gram of hydroxyapatite. Measurement of the 4- 8 S 4i mutanase activity, effected in the manner to be described below, indicated that 0.48g of mutan was decomposed per gram of the bonded protein.

Reference Example II Measurement of the titer of immobilized hydroxyapatite A precisely weighed amount of each sample (lmS of the undried sample before freeze-driying for immobilized hydroxyapatite and 2g of the sample for dentifrice 2 was added to 10m of a potassium phosphate buffer solution )1 containing 1% of each substrate and having a PH value of and a concentration of 0.05 mole, and the resulting solution was stirred at 35" for 2hr, followed by centrifugation. The residue was washed with the same buffer solution, and was combined with the centrifugate to determine the amount of each decomposition product. In the case of dextranase, the substrate was dextran, and the S0' amount of the decomposition product glucose was measured Sby the glucose oxidase method. In the case of mutanase, the substrate was mutan, and the amount of the decomposition product glucose was measured by the same method. In the case of levanase, the substrate was levan, and the amount of the decomposition product fluctose was determined by high performance liquid-chromatography.

Reference Exmaple III The following are thb results of measuring the 1 temporal change in the enzymatic activity of the dentifrice

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4 -9- 3 d r I i) sample prepared in Exmaple 1,2 and 3.

change in specific activity with time at 37° and PH (activity value just after preparation is taken as being 100) Ex. 1 Ex. 2 Ex. 3 100 100 100 154.0 155.0 327.0 40days 84.7 85.3 185.5 60days 70.5 68.4 125.4 80days lOOdays 60.3 52.2 55.4 46.4 96.4 84.3 010 r 0

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It was evident that the dentifrice containing the immobilized hydroxyapatite obtained by the present method exhibits an increase in activity from just after preparation and reaches the peak activity value after the passage of a certain period of time, following which there is a gradual decline in activity. However, the dentifrice maintained a higher enzymatic activity over an extended period, as compared with its initial activity.

The dentifrice composition according to the present invention comprise hydroxyapatite in which enzymes such as levanase, dextranase and mutanase useful for the prevention of dental caries are immobilized. It dispenses with any stabilizer and excels in stability, unlike the conventional enzyme containing dentifrice. The dentifrice composition of the present invention is also advantageous in that it can be prepared by using the immobilized IO-:a 10 L .l.J i\ i hydroxyapatite obtained in the present invention in place of the conventional polyshing agent in the conventional manner without recourse to any special procedure,.

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Claims (6)

1. A dentifrice composition containing hydroxyapatite having a glucanase or the like immobilized therein.

2. The dentifrice composition according to claim 1 wherein the glucanase is levanase.

3. The dentifrice composition according to claim 1 wherein the glucanase is dextranase.

4. The dentifrice composition according to claim 1 wherein the glucanase is mutanase.

A dentifrice composition containing hydroxyapatite having a glucanase or the like immobilized therein, substantially as hereinbefore described with reference to any one of Examples 1 to 5 or Reference Example 3.

6. A method of preventing dental caries in a patient, comprising administering to the oral cavity of the patient an effective amount of a dentifrice composition according to any one of claims 1 to DATED this FIRST day of MAY 1990 Dental Kagaku Kabushiki Kaisha Cr C.) SC U T Patent Attorneys for the Applicant SPRUSON FERGUSON ft S S S: S 555 I :1 c 4i"l I: t 6: I: 4 -4 S S S t 5 1 TMS/911c 4