An enzyme-containing dentifrice composition and its manufacture

The invention relates to a novel enzyme-containing dentifrice and to a process for the manufacture thereof.

Tooth decay, especially caries, is caused by a number of factors. It is common knowledge that as a rule there is a deposit upon the surface of a tooth consisting of food products and bacteria, which deposit is called plaque. The bacteria occurring in this plaque cause the food products to decay, during which process acids are formed. At the surface of the tooth the pH of the saliva, which generally has a value of 7.0 - 7.5, falls through the said formation of acids. Dependent upon the nature of the food consumed and the frequency of consumption, the pH can reach varying values, while the time required for the reaching of the normal pH (regeneration time) can also vary greatly. Thus, for example, it has been found that during the consumption of sugars in the form of tough masses, such as toffees, a much lower pH is reached than when these sugars occur in a product with a fibrous structure, such as apples, the regeneration time in the first case being moreover much longer. Further it has been found that the repeated consumption of sweets or other products forming acids, the regeneration time gradually increases. Dependent upon the nature of the material the pH can reach values of from 5.5 to 4.5 and sometimes even lower. The zone below the limit of pH 5.5 is often called the danger zone because under such acid conditions the calcium compounds of the tooth will dissolve in the saliva leading to decay of the tooth. Consequently it will be clear that the lower the pH is and the longer the regeneration time, the greater will be the risk of the teeth being

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affected.

Besides these two factors there is at least one other factor which plays an important part in tooth decay, i.e. the thickness of the plaque. The fact of the matter is that if the plaque is very thick the acids formed in it by bacterial decomposition have great difficulty in diffusing to the surface, resulting in a lower pH and a longer regeneration period.

In connection with last-mentioned factor it has been conventional for a considerable time past to incorporate abrasive and/or polishing material in tooth-pastes for the purpose of removing or reducing the plaque, which is the reason why the eating of apples is recommended.

It is also known to incorporate enzymes in tooth-pastes and similar dentifrices, e.g. proteases, such as pepsin, pancreatin, 15׳ trypsin and the' like־, or amylases. All the enzymes that have been applied so far belong to the group of hydrolases, so to the enzymes which are i.a. capable of breaking peptide or glycoside bonds in consequence of which macro-molecular compounds are converted into oligo- or. monomer products which dissolve more easily in saliva and can thus be removed

From United States Patent 2,035,896 it was known to incorporate catalase and a perborate in tooth powder. The function of the enzyme ; is to liberate hydrogen peroxide from the perborate. Because of the decomposition of hydrogen peroxide no inactivation of the bacteria in the mouth takes place.

According to the dentifrices according to the invention an oxidoreductase yielding hydrogen peroxide is incorporated into the tooth powder, if necessary, together with its substrate. Because of the hydrogen peroxide which is slowly and continuously formed inactivation of the mouth flora takes place indeed preventing the formation of harmful acids.

a The present Invention is thus based on an entirely different and new principle consisting in that to a dentifrice an oxido-reductase is added which will form hydrogen peroxide in the decomposition of the substrate. By using such enzyme systems in dentifrices two important effects are reaced: the plaque is affected and, what is of special importance, hydrogen peroxide is formed in the enzymatic decomposition, which has been found to normalize the mouth flora, in consequence of which a decrease is seen in bacterial decomposition ,and, consequently, no or hardly any formation of noxious acids.

In principle any oxidoreductase enzyme supplying hydrogen peroxide can be used in the manufacture of the preparations according to the invention, in general the oxidoreductases which decompose the substrate with oxygen as the acceptor.

As examples of such enzymes can be mentioned the oxidases acting upon the CIIOH-, aldehyde- or keto group of the substrate, such as lactate oxidase, malate oxidase, glucose oxidase, hexose oxidase, galactose oxidase, pyruvate oxidase and oxalate oxidase, oxidases acting upon the CH-NH group of the substrate, such as L-amino-acid oxidase, D-amino-acid oxidase, tyraminase and histaminase, and further oxidases which have another point of attack, such as xanthine oxidase, sarcosine oxidase, N-methylamino-acid oxidase and sulfite oxidase.

The quantity to be used of the enzyme mentioned before can vary greatly, but it is usually between 0.01 and JOO U per gram or millilitre of the dentifrice.

By one unit of enzyme is meant that quantity of enzyme which will oxidize 1 μ mol substrate per minute at JO°C and pH 6 under standard conditions.

It has proved to be advantageous also to add one or more other enzymes, especially those enzymes which can supply substrate for the oxidase enzyme system applied, such as enzymes belonging to the group of hydrolases. The hydrolases can be for example carbohydrases, proteases and lipases such as a-amylase, β-amylase, glucoamylase or amyloglucosidase, cellulase, dextranase, invertase, a- and βglucosidase, a- and β-galactosidase, pepsin, trypsin, chymotrypsin, papain and proteases of bacterial origin.

These hydrolases are measured according to conventional methods, and their activities are indicated in units in accordance with the recommendations of the International Union of Biochemistry (Report of the Commission in Enzymes of the IUB, Pergamon Press, Oxford, 1961).

Instead of, or simultaneously with one or more hydrolases it is also possible to add to the preparation the substrate of the oxidase to be applied, such as a sugar, for example galactose or glucose, a lactate or malate, an amino acid, a pyruvate or oxalate, and the like.

The addition of glucoseoxidase together with amyloglucosidase has also proved to be a very favourable combination.

In order to show the surprising effect of the said enzymes the pH-valuc was measured at the surface of the tooth in the plaque. The micro-antimony electrode used for this purpose has been described in the literature by F. Clarence Thompson et al. in Journal of Dental Res. 33 (1954), 849. The measurings were performed on the buccal surfaces of the first and second molar, at least one hour after the last consumption of food. So there were 4 measuring points in total per person, always in the sequence: M^SD-M2SD-M^SS-M2SS.

On the first day they rinsed their mouth for 4 minutes with 10 ml of a ?0% saccharose solution, after which the pH was measured. After these measurings they rinsed with 10 ml of mouth wash in which the enzymes had been dissolved, which, of course, was not done by the controls.

The tests were performed so that there was an interval of at least 1 hour between the last rinse and the next meal.

On the second day the process was repeated with 10 ml of a ?0% saccharose solution, after which the pH was measured.

The next day the useful effect of the rinse with enzyme on the pH could be clearly demonstrated by measuring the pH at the surface of the tooth after the rinse with saccharose.

In order to avoid mechanical effect of the plaque both the saccharose and the enzymes were applied in a liquid form.

The tests were performed on groups of 15 - 30 children (boys and girls) of from 12 to 14 years.

In the following table the results are .given of the pH measurings.

The children have been divided into 3 groups:

a. those with a pH higher than 6.8

b. those with a pH between 5.6 and 6.8

c. those with a pH lower than 5.6.

<td rowspan="2"></td><td colspan="3"> Percentage of children with pH</td>

<td> &gt; 6.8</td><td> 5.6 - 6.8</td><td> &lt; 5.6</td>

<td> Controls 1st day 2nd day</td><td> 23 19</td><td> 53 58</td><td>־ γλ &lt;\J r.j</td>

<td> JO U amyloglucosidase per 10 ml 1st day 2nd day</td><td> 33 28</td><td> 39 47</td><td> 28 25</td>

<td> glucose oxidase 6 U $i,U£J1aliisUS£ per 10 ml 1st day 2nd day</td><td> 25 31</td><td> 55 60</td><td> 20 Q</td>

<td> JO U amyloglucosidase + 6 U glucoseoxidase per 10 ml 1st day 2nd day</td><td> 21 60</td><td> 59 40</td><td> 20 0</td>

From this table it appears that amyloglucosidase only has no appreciable influence on the percentage of children in the danger zone below 5.6, and that glucoseoxidase, and to an even higher degree glucoceoxidase in combination with amyloglucosidase reduces this percentage considerably or to zero.

Another striking factor was that the structure of the plaque had become less firm by the treatment with the enzyme mixture so that the plaque came off easily in maty cases.

The dentifrices according to the invention can occur in various forms, e.g. in the form of tooth-paste, mouth wash, tablets, chewing-gum or other conventional forms. Besides the enzyme products according to the invention and a substrate, if any, for the oxidase applied these dentifrices contain the conventional substances. Thus, for example, in tooth paste are incorporated abrasive and/or polishing materials such as calcium carbonate, dicalcium phosphate, calcium phosphate, calcium sulphate, or silicium compounds, thickeners such as carboxymethylcellulose, tragacanth or guar, water, flavourings and/or natural or synthetic sweetening agents. Further fluoro compounds can be added such as sodium- or potassium-monofluoro phosphate or sodium fluoride.

The invention is illustrated further by the following compositions,, to which it is, of course, not limited.

Example

Too;h-paste

Precipitated silica (e.g. Neosyl) about 23 %

Paraffin 15 %

PAB-esters 0.2 %

Methylcellulose 1.8 %

Aromatic substances 2 %

Glucoseoxidase (20 U/gm)

Amyloglucosidase (30 U/gm)

Distilled water up to 100 %

<td> •</td><td> Example</td><td> II</td>

<td></td><td> Tooth-paste</td><td></td>

<td></td><td> Calcium carbonate</td><td> 50 %</td>

<td></td><td> Tricalcium phosphate</td><td> 5 %</td>

<td> 5</td><td> Sorbitol (70% solution)</td><td> 10 %</td>

<td></td><td> Glycerol</td><td> 20 %</td>

<td></td><td> . Tragacanth</td><td> 2 %</td>

<td></td><td> Aromatic substances</td><td> 0.8 %</td>

<td></td><td> Glucoseoxidase (10 U/gm)</td><td></td>

<td> 10</td><td> Dextranase (10 U/gm)</td><td></td>

<td></td><td> PAB-esters</td><td> 0.1 %</td>

<td></td><td> Distilled water</td><td> up to 100 %</td>

<td></td><td> Example</td><td> III</td>

<td> 15 -</td><td> Tooth-paste</td><td></td>

<td></td><td> Aluminium-hydroxide</td><td> 40 %</td>

<td></td><td> Na-fluoride</td><td> 0.1 %</td>

<td></td><td> Sorbitol (70% solution)</td><td> 25 %</td>

<td></td><td> Glycerol</td><td> 5 %</td>

<td> 20</td><td> Aromatic substances</td><td> 1.2 %</td>

<td></td><td> Na-alginate</td><td> 1 %</td>

<td></td><td> PAB-esters</td><td> 0.1 %</td>

<td></td><td> Saccharine</td><td> 0.25%</td>

<td></td><td> Glucoseoxidase (5 U/gm)</td><td></td>

<td> 25</td><td> Invertase (25 U/gm)</td><td></td>

<td></td><td> Water</td><td> up to 100 %</td>

Example

Tooth-powder

Aromatic substances 2 %

Na-cyclamate 0.5 %

Detergent (Texapon L 100) 1 %

Galactoseoxidase (4 U/gm)

Glucoseoxidase (4 U/gm)

P-galactosidase (30 U/gm)

Calcium phosphate up to 100 %

Example

Mouth wash

Methylcellulose (low viscous) 1 %

Aromatic substances 1 %

PAB-esters 0.15%

Dinatrium phosphate 0 aq. 1.5 %

Citric acid 1 aq. 1.0 %

Glucoseoxidase (3 U/ml)

Amyloglucosidase5)'־ U/ml)

Distilled water up to 100 %

Example

Tooth-powder

<td> Na-cyclamate</td><td> 0.75%</td>

<td> Aromatic substances</td><td> 2.25%</td>

<td> L-amino acid oxidase (95 U/gm)</td><td></td>

Example

Glycine

Tricalcium phosphate up to 100 %

<td rowspan="2"> 5</td><td colspan="3"> Tooth-powder</td>

<td> Aromatic substances</td><td> 1</td><td> %</td>

<td></td><td> Na-fluoride</td><td> 0.1</td><td> %</td>

<td></td><td> Detergent</td><td> 1</td><td> %</td>

<td></td><td> Xanthine</td><td> 0.8</td><td> %</td>

<td> 10</td><td> Xanthineoxidase (5 U/gm)</td><td></td><td></td>

<td></td><td> Na-saccharine</td><td colspan="2"> 0.25%</td>

<td></td><td> Micro-crystalline aluminium hydroxide up to</td><td> 100</td><td> %</td>

Example VIII

<td> 15</td><td colspan="4"> Chewing tablet</td>

<td></td><td> Carbowax 6000</td><td></td><td> 2</td><td> %</td>

<td></td><td> Aromatic substances (micro-capsules)</td><td></td><td> 0.5</td><td> %</td>

<td></td><td> Colouring matter Glucoseoxidase (5 U/gm)</td><td></td><td> 0.1</td><td> %</td>

<td> 20</td><td> Pancreatin</td><td></td><td> 1</td><td> %</td>

<td></td><td> Mannitol Example Chewing-gum</td><td> up to</td><td> 100</td><td> %</td>

<td></td><td> Gum basis</td><td></td><td> 14</td><td> Λ’</td>

<td> 25</td><td> Sorbo (?0%)</td><td></td><td> 25</td><td> %</td>

<td></td><td> Aromatic substances Glucoseoxidase (50 U/gm) Invertase (100 U/gm)</td><td></td><td> 0.5</td><td> %</td>

<td></td><td> Sorbitol</td><td> up to</td><td> 100</td><td> %.</td>