FR2519552A1 - Stabilisation of lysozyme - by incorporation with sugar or glycerine and a plasticising agent - Google Patents

Abstract

Lysozyme (I) is stabilised in liquid or semi-liquid compositions by incorporation in sugar or glycerin previously diluted in a solvent and in the presence of one or more non-ionic or anionic plasticisers. After mixing, excipients are also incorporated. (I) may be used as such, or as salt, pref., the hydrochloride, chloride, or carbonate. The glycerine may be pure, or one of its derivatives may be used. The sugar may be a mono-di-, or polysaccharide, an amino sugar or an alcohol derivative such as sorbitol or mannitol. Suitable plasticisers may be cellulosic, an alginate, or a resin such as a polyoxyethylene resin. (I) is an antimicrobial and antiviral agent. The compositions are useful as dentifrices, ointments for the treatment of disorders causing blistering of the skin and mucous membranes, and mouthwash solutions.

Description

The present invention relates to an improved process for stabilizing lysozyme in a liquid or semi-liquid medium, as well as a united composition based on lysozyme, and more particularly a stable composition containing lysozyme, usable in therapy and in hygiene.

We know that lysozyme is a protein of natural origin, generally extracted from chicken egg white, having anti-microbial and antiviral properties allowing its application in therapy, especially for the treatment of conditions such as apthoses, l herpes, zonas, gingivitis, and more generally most vesicular and bullous diseases of viral origin. Lysozyme acts in particular on tooth decay by lowering the production of acids by bacteria in dental plaque.

However, the use of lysozyme proves difficult in practice, because of its great instability in an aqueous medium or in the presence of certain compounds such as carbonates, alginates, salts of heavy metals, etc., commonly used as excipients, plasticizers, or carriers of various drug compositions.

In these conditions, the lysozyme very quickly loses all enzymatic action, for example in solution in water, or else there is an adsorption which causes an immediate drop in its action, which can be irreversible depending on the compounds concerned. As a result, antimicrobial and antiviral actions are greatly limited and may even disappear completely in some cases.

The present invention relates to a process for stabilizing lysozyme in a liquid or semi-liquid composition.

The invention also relates to a composition based on lysozyme avoiding the drawbacks mentioned above.

A more particular subject of the invention is a composition based on lysozyme, which can be used in therapy, providing excellent storage stability.

The process according to the present invention making it possible to stabilize lysozyme, pure or in the form of a salt, in a liquid or semi-liquid composition, consists in incorporating the lysozyme in a sugar or in a glycerin, previously diluted in an appropriate solvent, presence of one or more nonionic or anionic plasticizers, to be mixed then to gradually add the excipients and, if necessary, the associated active ingredients.

According to a preferred embodiment of the invention, a sugar such as sucrose or glucose is diluted in water, one or more nonionic or anionic plasticizers are added, then lysozyme, pure or in the form of hydrochloride, and the glycerin is poured into solution in an alcohol, if necessary an additional active principle is added, and finally the usual excipients are incorporated and mixed.

In accordance with the present invention, it is particularly advantageous to dilute the sugar in water, at room temperature, to add one or more nonionic or anionic plasticizers, then to incorporate glycerin and if necessary an additional amount. sugar, then add the lysozyme.

The lysozyme composition in accordance with the present invention contains lysozyme in the pure state or in the form of a salt, in combination with one or more sugars and / or glycerin or a glycerin derivative, and if appropriate one or more usual excipients or other active ingredients.

According to a preferred form of implementation of the invention, lysozyme, pure or in the form of a salt, is associated with a glycerin and a sugar, the combination of these three components giving the composition even more improved stability compared to a composition. containing lysozyme and a sugar, or lysozyme and a glycerin.

It is particularly advantageous, according to the invention, to additionally incorporate a nonionic or anionic plasticizer such as a cellulose derivative, an alginate such as sodium or potassium alginate, or a resin, and for example a resin polyoxyethylene.

Lysozyme is used pure (isoelectric lysozyme), or in the form of a salt, and preferably in the form of the hydrochloride, or, where appropriate, in the form of lysozyme chloride or carbonate.

The glycerin is preferably pure glycerin (or glycerol), but it can optionally be replaced by one of its usual derivatives.

The sugar is preferably chosen from monosaccharides such as glucose or fructose, disaccharides such as sucrose, des.

polysaccharides or also derivatives, and in particular amino derivatives such as glucosamine, or alcoholic derivatives such as sorbitol and mannitol. Sugar is used alone or as a mixture of several sugars.

In the composition according to the present invention, the lysozyme can be present in an amount of 10 μg to 0.59, and preferably 30 μg to 3 mg per gram of composition, while the weight ratio of the other components is between 5 and 30%, and preferably between 15 and 20% for sugar, and between 1 and 20%, and preferably between 2 and 10 for glycerin, the rest being constituted by the usual plasticizers and excipients such as alginates, phosphates , biopolymers, etc., and where appropriate, other associated active ingredients, such as vitamins, local anesthetics, antibiotics, antifungals, etc.

The pH of the composition is maintained at a value between 4.5 and 7.5 by a usual buffer, such as a phosphate buffer.

The examples given below illustrate the invention without limiting its scope. In these examples, the ratios, proportions and percentages are given by weight, unless otherwise indicated.

EXAMPLE 1
In an aqueous solution of 100 ml of phosphate buffer with a pH of 6.2, 1 g of lysozyme hydrochloride is added with stirring.

In this solution, a solution of 1.49 glycerin in a minimum quantity of ethyl alcohol is added with stirring; mixed thoroughly, then 49 sodium alginate and 0.29 methyl p-hydroxybenzoate are added.

A stabilized composition of lysozyme is thus obtained, in the form of the hydrochloride, containing glycerin.

EXAMPLE 2
The procedure is as indicated in Example 1, but 109 of sucrose is added to the buffered aqueous solution of lysozyme hydrochloride.

The glycerin solution is then added as in Example 1.

This gives a lysozyme hydrochloride solution stabilized by the addition of sucrose and glycerin.

EXAMPLE 3
By proceeding as indicated above, but by replacing the sucrose by a mixture of sorbitol and sucrose, and by adding vitamin A at the same time as the glycerine, a composition based on lysozyme is obtained, having the following formulation, usable as an ointment.

Lysozyme hydrochloride 0.3 g
Sorbitol 10.0 g
Sucrose 10.0 g
Glycerin 1.4 g
Sodium alginate 4.0 g
Zinc oxide 2.0 g
0.2 g methyl p-hydroxybenzoate
Vitamin A 20x106u.i.

water qs 100 9
EXAMPLE 4
By proceeding as indicated previously, a toothpaste having the composition below is prepared:
Lysozyme hydrochloride 25 mg
Sorbitol 7.5 g
Sucrose 7.5 g
Glycerin 10.0 g
Paraffin oil 0.5 g
Dicalcium phosphate dihydrate 38.0 g
Carboxymethylcellulose sodium 0.259
Sodium alginate 1.759
Sodium methoxide 0.1 g
Water qs 1009
EXAMPLE 5
A toothpaste is prepared, by operating as indicated below, for a final quantity of paste of 1009.

2.79 of sorbitol and 0.3 g of sodium saccharinate are dissolved in 20 ml of deionized water at room temperature, and 0.1 g of methyl p-hydroxybenzoate and 0.1 g of propyl p-hydroxybenzoate are added.

While stirring the solution, add 29 of sodium carboxymethylcellulose and allow to stand overnight.

Then pour a solution of 15 ml of deionized water containing 7.79 sucrose, 59 sorbitol, 10.19 glycerin, 40.59 dicalcium phosphate dihydrate and 0.59 paraffin oil, and pour this solution in a mixture of 25 mg of lysozyme hydrochloride, O, lg of sucrose and O, lg of glycerin in lml of deionized water. This mixture is poured slowly with stirring at room temperature.

0.99 lauryl sarcosinate sodium and the usual aromatic components are added to this mixture 0.99. Mix thoroughly with stirring for approximately 30 minutes.

As indicated above, the compositions in accordance with the invention exhibit excellent stability, even in the presence of a liquid such as water or ethanol which react with the lysozyme and destroy its activity or mineral salts such as sodium carbonate or alginate which inhibit the activity of lysozyme by adsorption.

More particularly, the lysozyme-based compositions in accordance with the invention described above remain stable for a period of approximately 2 years, that is to say that the activity of lysozyme is fully preserved during this period. . The activity then decreases slowly and regularly and after 3 years there is still an enzymatic activity equal to approximately half of the initial activity. By way of comparison, the usual lysozyme hydrochloride solutions, generally prepared extemporaneously by dissolving in an aqueous phosphate buffer solution of pH 6.2, have a stability limited to a few hours.

The lysozyme-based compositions in accordance with the invention can be used more particularly for the preparation of toothpastes, ointments for the treatment of vesicular and bullous diseases of the skin and mucous membranes, and solutions for mouthwashes.

Claims (10)

1. A method for stabilizing lysozyme in a liquid or semi-liquid composition, characterized in that the lysozyme is incorporated in a sugar or glycerin previously diluted in a solvent, in the presence of one or more nonionic or anionic plasticizers, it is mixed, and then the excipients are added. 2. Method according to claim 1, characterized in that a sugar is diluted in water, one or more nonionic or anionic plasticizers are added, then lysozyme, and glycerin is poured in solution in an alcohol, and the excipients are incorporated where appropriate. 3. Method according to claim 1, characterized in that the sugar is diluted in water, one or more non-ionic or anionic plasticizers are added, and the glycerin and then an additional amount of sugar are incorporated, then add the lysozyme. 4. Method according to claim 3, characterized in that the lysozyme is added in the form of a solution in deionized water containing sugar and glycerin. 5. Method according to any one of claims 1 to 3, characterized in that the plasticizer is an alginate or a biopolymer. 6. Composition based on lysozyme usable in therapy and in hygiene, characterized in that it contains lysozyme in the pure state or in the form of salt, in combination with one or more sugars and / or glycerin or a derivative of glycerin, one or more nonionic or anionic plasticizers, and if necessary one or more other active ingredients and excipients. 7. Composition based on lysozyme according to claim 6, characterized in that it contains lysozyme, one or more sugars and glycerin. 8. Composition based on lysozyme according to any one of claims 6 and 7, characterized in that the sugar is chosen from glucose, fructose, sucrose, glucosamime, sorbitol and mannitol. 9. Composition based on lysozyme according to claim 7, characterized in that it contains lysozyme, glycerin, sucrose and / or sorbitol. 10. Composition based on lysozyme according to claim 6, characterized in that the lysozyme is in the form of the hydrochloride.