GB2164052A - Polymeric material having an enzymatic action, its process of preparation and a medicament containing it - Google Patents

Abstract

A polymeric material having an enzymatic action comprises a water-swellable hydrophilic carrier of crosslinked poly- beta -alanine carrying aldehyde groups, to which at least one proteolytic enzyme is fixed by covalent bonding. This material is useful as a medicament for cleansing wounds and assisting their cicatrisation, or as a cosmetic composition.

Description

SPECIFICATION Polymeric material having an enzymatic action, its process of preparation and a medicament containing it The present invention relates to a novel polymeric material having an enzymatic action, its process of preparation and its use, particularly as a medicament for the treatment of wounds for the purpose of cleansing and cicatrising them.

The cleansing of wounds, burns, ulcers etc. is currently effected by enzymatic cleansing agents which act upon the exudates and the necrotic products.

Among the enzymatic cleansing agents currently used, the products based on trypsin, chymotrypsin or papain, optionally associated with other active principles, may be mentioned.

These products permit good cleansing, a factor which activates cicatrisation. However, they may cause intolerance phenomena due essentially to an allergy to the enzymatic proteins employed.

Furthermore, the enzymes generally used in these cleansing products lack heat stability, particularly at the temperatures at which they are used, resulting in an appreciable loss of activity not only during storage but also when the products are applied to the skin.

It has now been found that the disadvantages of these enzymotherapeutic cleansing agents can be largely overcome by fixing the enzymes to a polymeric substrate by covalent bonding.

Indeed, the enzyme fixed on the polymer is absorbed little, if at all, at the wound, and the allergic phenomena is then reduced or even eliminated.

It has also been observed that the enzymes thus fixed exhibited good stability of activity, substantially greater than that of the free enzymes at temperatures of from 35 to 50"C.

The reduction in the decline of enzymatic activity with time permits an appreciable reduction in the quantity of enzymatic proteins usually employed in the known enzyme-based cleansing agents.

The present invention therefore provides a polymeric material capable of enzymatic action, which comprises a hydrophilic water-swellable carrier of cross-linked poly-fi-alanine carrying aldehyde groups, to which is covalent bonded at least one proteolytic enzyme.

The polymeric substrate can be obtained by cross-linking poly-ss-alanine by means of a crosslinking agent, particularly by means of a dialdehyde, such as glutaraldehyde. Poly-ss-alanine is a known polymer, the preparation of which has been described in, for example, US Patents Nos.

2,749,331 and 4,082,730.

The crosslinking is generally performed in a suspension of the aqueous solution of poly-fi- alanine in an organic solvent such as cyclohexane, toluene, methyl benzoate, benzyl benzoate, chlorobenzene or dichloroethane in the presence of a suspension agent such as cellulose derivatives, particularly ethylcellulose, ethylhydroxyethylcellulose and hydroxyethylcellulose, polyvinyl acetate, a maleic anhydride octadecyl vinyl ether copolymer, a maleic anhydride octadecene copolymer, sorbitan oleates and the condensation products of ethylene oxide and propylene oxide known under the trade name "PLURONIC".

The essential purpose of the suspension agent such as mentioned above is to direct the formation of cross-linked poly-ss-alanine into the form of spheres, the diameter of which is a function of the type and proportion of the suspension agent used.

Thus, when polyvinyl acetate is used as the suspension agent, the spheres of crosslinked poly-ss-alanine generally have a greater diameter than those obtained when "Pluronic L84" or hydroxyethylcellulose is used as suspension agent.

The proportion of the suspension agent also plays an important part in obtaining spheres of the desired diameter. It may vary within wide proportions but is generally from 0.1 to 5% by weight.

The amount of the crosslinking agent, eg. dialdehyde, may vary within wide proportions but is generally from 1 to 20% by weight relative to the weight of the water-soluble poly-fi-alanine reacted.

The crosslinking reaction is suitably conducted at a temperature of from 20 to 800C and at an acid pH, preferably from 1 to 2; obtained by the addition of an inorganic acid, for example hydrochloric acid.

After agitation at-ambient temperature, the reaction mixture is typically heated to a temperature of from 40 to 100"C for, say, 1 to 6 hours.

The spheres obtained are then separated off, thereafter washed with an organic solvent or an alcohol/caustic soda mixture, then with water and optionally with ethanol, and finally oven-dried.

The diameter of the spheres of crosslinked poly-ss-alanine generally vary from 2 to 500 microns, as a function of the type and quantity of the suspension agent employed, and also of the operating conditions, such as, for example, the speed of agitation.

The spheres obtained may then be subjected to a screening operation in order to separate off those with a diameter which is smaller or greater than a certain value, for example outside the range of from 100 to 200 microns.

By the process as described above, the spheres obtained are capable of swelling in water by 2 to 25 times their initial volume, depending on the degree of crosslinking. Moreover, depending on the proportion of dialdehyde used relative to the initial polyalanine, the the spheres obtained may contain a higher or lower proportion of free aldehyde groups which have not taken part in the crosslinking reaction.

These aldehyde groups, which should be small in number when it is desired to use the crosslinked poly-fi-alanine as a cleansing agent by absorption, are on the other hani necessary for the fixation of enzymes by covalent bonding.

In fact, the enzymes which carry primary amine groups can react with the aldehyde groups of the substrate by a reaction of the Schiff's base type, thus fixing the enzymes by covalent bonding.

Among the different enzymes capable of being fixed by covalent bonding to the substrate of cross-linked poly-fi-alanine, the following may be specially mentioned: -the non-specific proteolytic enzymes (with a broad spectrum of action) such as %-chymo- trypsin, trypsin, papain, fibrinolysin, streptokinase, streptodornase (streptococcus haemolyticus), and bacterial (Bacillus subtilis) or fungal (Aspergillus orizae) proteolytic extracts, -or the more specific proteolytic enzymes (with a narrow spectrum of action) such as collagenase, the activity of which is limited to natural and denatured collagens.

In the fixation reaction which will be described below, it is preferable to use a large excess.of the enzyme which is desired to fix, so as to prevent free aldehyde groups remaining, which might have a harmful influence, particularly causing irritation phenomena.

The process of fixing the enzymes by covalent bonding consists in placing the spheres of crosslinked poly-fi-alanine in a buffer solution of pH from 6 to 9.5, preferably from pH 8.5 tor 9.2, or at a pH appropriate to the type of enzyme. The spheres, on contact with the buffer solution, swell and pass into suspension.

The mixture is then agitated for approximately 1 to 24 hours in an apparatus permitting the supply and discharge of the buffer solution at a constant flow rate. The resulting gel is then filtered.

The enzyme is then dissolved at approximately 4"C in a buffer solution identical to that used for the swelling of the crosslinked poly-ss-alanine, to which solution the gel obtained previously is added.

The mixture is agitated in a nitrogen atmosphere for approximately 1- to 48 hours, and preferably for 3 to 30 hours, at approximately 4"C.

The gel carrying the enzyme is then filtered off on a glass frit and is resuspended in distilled water at approximately 4"C, filtered again and washed repeatedly until the wash waters exhibit no further detectable enzymatic activity.

The Uspheres obtained are then dried, preferably by freeze-drying, and thereafter screened.

The contact time necessary to fix the enzymatic proteins depends, at 4"C, on the type of protein on the one hand, and on the operative pH on the other hand.

The polymeric material is preferably sterilised by any conventional method, particularly by irradiation, before its use as a medicament.

The present invention also provides, by way of a medicament for the treatment of wounds, particularly for cleansing and cicatrising them, which cotnprises, in a vehicle suitable for local application the novel polymeric material having an enzymatic action as defined above, either alone or in the form of a mixture with another polymeric material, the fixed enzyme of which is a different type.

The medicament may be used as it is or in the form of a powder, the particle sizes of which are generally from 100 to 250 microns. This powder may be applied directly onto the wounds after these have been washed in water or with physiological serum.

The medicament may also assume the form of an ointment containing, in a suitable pharmaceutical excipient, the spheres in a proportion corresponding to the required enzymatic activity.

In addition to the various advantages which have been mentioned previously, the medicament permits the cleansing to be effected not only by enzymatic means but also by absorption, the latter resulting from the very nature of the hydrophilic support, which has the property of absorbing the liquids from the wounds, thus facilitating the cicatrisation process.

The medicament therefore makes it possible to effect the cleansing and the cicatrisation of wounds by a double action, that is to say by enzymotherapy and by absorption.

According to a particular embodiment, the medicament may likewise contain, together, an active principle such as an antibiotic like neomycin, a local anaesthetic such as lidocaine hydrochloride (DCI) or butoform (DCI), an antiseptic such as 3,4,4'-trichlorocarbanilide or triclocarban (DCI), an antifungal agent such as miconazol (DCI) or an anti-inflammatory agent such as hydrocortisone.

The medicament is recommended most particularly for the treatment not only of chronic, atonic, and ulcerated lesions but also ulcers of vascular origin, varicose ulcers, post-phlebitic ulcers and arteritic ulcers, in the treatment of burns and of bedsores and more generally in relation to any wounds which exhibit delay in cicatrisation.

Although reference has been made more particularly to the use of the polymeric material having an enzymatic action as a medicament, it is self-evident that it may likewise be used as an active ingredient in cosmetic or dermatological compositions.

A number of examples of preparations of the medicament and also a number of examples of application, will now be given by way of illustration.

EXAMPLE 1: Fixing of trypsin at pH 9.2 a) Preparation of crosslinked poly-fi-alanine in the form of spheres.

Into a 2-litre reaction flask fitted with a nitrogen inlet, condenser, thermometer, dropping funnel and propeller stirrer, 600 g of distilled cyclohexane are introduced, followed by 3 g of ethylcellulose T 100, (sold by Messrs. HERCULES), which is dissolved by heating with stirring, under nitrogen.

After cooling to 25"C, the stirring speed is adjusted to 1300 rpm and the following are introduced under nitrogen in 15 min: an aqueous solution of poly-ss-alanine and glutaraldehyde obtained by dissolving 100 g of poly-ss-alanine in 70 cc of water previously degassed and saturated with nitrogen, then 1 g of ascorbic acid and 30 g of a 25% strength aqueous solution of distilled glutaraldehyde, and sufficient concentrated (d=1.18) hydrochloric acid in order to bring the pH to 1.

The addition being complete, the mixture is stirred for 10 min at ambient temperature and then heated to 50"C for 3h 30 min. After this reaction time the mixture is filtered on a Buchner funnei.

The spheres of crosslinked poly-ss-alanine obtained are then subjected to the following series of washings: (i) suspension in 800 g of cyclohexane for 15 min with solvent reflux, followed by filtration, (ii) suspension in 800 g of pure ethanol, followed by filtration, (iii) suspension in water (800 ml), followed by filtration, (this operation is repeated 3 times until the last aqueous phase is neutral) and (iv) suspension in pure ethanol, followed by final filtration.

The moist spheres are then dried on trays lined with filter paper for 24 h in ambient air and thereafter for 24 h in an oven at 40"C.

The spheres are then screened on two sieves with 250 and 100 micron meshes, producing 50 g of a yellowish white powder of the required size.

Measurement of swelling: A 10-ml test tube is filled with 1 ml of spheres in the dry state, the diameter of which is between 100 and 250 microns. This is then made up to 10 ml by adding water; the spheres swell rapidly up to 5.7 ml (in the absence of any agitation).

(b) Preparation of the crosslinked poly-,-alanine carrier at pH 9.2.

4 g of crosslinked poly-ss-alanine in the form of spheres obtained above under a) are placed in 80 ml of a buffer solution of sodium borate (19.7 g of Na2B407.10H20 in 1 litre of water). The spheres swell rapidly and the mixture is stirred for 1 h in an apparatus permitting the supply and discharge of the buffer solution at a constant flow rate of 60 ml/h. The gel is then filtered off on a sintered glass filter, the internal pressure of which is 30 mm Hg.

c) Fixation of the enzyme (trypsin) 477 mg of PROLABO trypsin assayed at 37,000 units/g are dissolved at 4"C in 48 ml of a solution buffered to pH 9.2, such as is used for the preparation of the carrier, and then the crosslinked poly-ss-alanine gel obtained under b) above is introduced. The mixture is left, with vigorous stirring, for 24 h at 4"C under a nitrogen atmosphere. The gel carrying the trypsin is transferred onto a glass frit, filtered off, then resuspended in 200 ml of distilled water at 4"C, and finally filtered off again. This operation is repeated until the wash waters exhibit no further trypsin activity; this necessitates at least 4 successive washings. The resulting polymer is dehydrated by freeze-drying.

Assay The activity is assayed by the method described in Biochemica Merck p. 48. The assay shows an activity of 1200 units/g.

EXAMPLE 2: Fixation of trypsin at pH 8.5 a) Preparation of the substrate poly-ss-alanine at pH 8.5 4 g of crosslinked poly-fi-alanine in the form of spheres prepared according to Example 1 a) are placed in 80 ml of a buffer solution (1000 ml of solution containing 0.025 M of sodium borate (Na2B407.10H20) and 152 ml of a 0.1 M solution of HCI). The spheres swell rapidly and the mixture is agitated for 1 hour in an apparatus permitting the supply and discharge of the buffer solution at a constant flow rate of 60 ml/h. The gel is then filtered off on a sintered glass filter, the internal pressure of which is 30 mm Hg.

b) Fixation of the enzyme (trypsin) 477 mg of PROLABO trypsin assayed at 37000 units/g are dissolved at 4"C in 48 ml of a solution buffered to pH 8.5, such as is used for the preparation of the substrate, and then the gel prepared under a) above is introduced. The mixture is left, with vigorous stirring for 6 hours at 4"C under a nitrogen atmosphere. The gel carrying the trypsin is transferred onto a glass frit, filtered off, then resuspended in 200 ml of distilled water at 4"C, and finally filtered again. This operation is repeated until the wash waters exhibit no further detectable trypsin activity (at least 4 washings). The resulting product is dehydrated by freeze-drying.

Assay The activity of the enzyme-polymer is assayed by the method described in Biochemica Mdrck p. 48. The assay shows an activity of 1800 units/g.

EXAMPLE 3: Fixation of a-chymotrypsin at pH 8.5 a) Preparation of the substrate poly-ssalanine 4 g of crosslinked poly-ss-alanine in the form of spheres prepared according to Example 1 a) are placed in 80 ml of buffer solution (1000 ml containing 0.025 M of sodium borate (Na2B407.10H20) and 152 ml of a 0.1 M solution of HCI). The spheres swell rapidly and the mixture is agitated for 1 hour in an apparatus permitting the supply and discharge of the buffer solution at a constant flow rate of 60 ml/h. The gel is then filtered off on a sintered glass filter, the internal pressure of which is 30 mm Hg.

b) Fixation of the enzyme (a-chymotrypsin) 500 mg of SIGMA a-chymotrypsin assayed at 52,500 units/g are dissolved in 4"C in 60 ml of the pH 8.5 buffer solution, as used for the preparation of the substrate, and then 5 g of crosslinked poly-fi-alanine in the form of a gel as obtained under a) above are introduced. The mixture is left for 24 hours with gentle stirring at 4"C. After this time, the gel supporting the achymotrypsin is filtered off on a glass frit, suspended at 4"C in 400 ml of distilled water and then filtered off again. This operation is repeated until the wash waters exhibit no further detectable a-chymotrypsin activity. The enzyme-polymer thus obtained is dehydrated by freezedrying.

Assay The activity of the enzyme-polymer is assayed according to the method of W. F. Nagel et al.

described in Hoppe Seyler's Z. Physiol. Chem. 1965, 340, 1. The assay shows an activity of 1464 units/g.

EXAMPLES OF APPLICATION A-Powder for external use Dusting bottle containing the enzyme-polymer obtained according to Example 1, at 1200 units/g, with a particle size of between 100 and 250 microns.

After having cleaned the wound by spraying with water or by means of physiological serum, a sufficient quantity of the enzyme-polymer is applied to it to cover the surface of the wound, and a compress is then applied in order to hold the dressing in place. On renewing the application once a day, rapid release of adhesions of the wound and acceleration of cicatrisation are found, without any irritation phenomena, such as those encountered when trypsin is used alone as a cleansing agent, being observed.

B-Powder spray for external use The following ingredients are packed in an aerosol can: -enzyme-polymer obtained according to Example 2, at 1800 units.g 9g -magnesium stearate 0.5 g -isopropyl myristate 0.2 g -difluorodichloromethane 54 g --1 ,2-dichlorotetrafluoroethane 36 g Before the powder is applied to a wound, the latter is first of all cleaned by means of phsiological serum, and a sufficient quantity of powder is applied to it to cover the surface of the wound, after which a compress is applied in order to hold the dressing in place.

C--Hydrophilic ointment -enzyme-polymer obtained according to Example 3, at 1464 units/g 7g -polyethylene glycol 400 60 g -polyethylene glycol 35/50 33 g This ointment is applied to a wound after having cleaned it by means of water or physiological serum, and a compress is then applied in order to hold the dressing in place.

The application of the ointment is repeated once per day, and an acceleration of cicatrisation is found, without any irritation phenomena being encountered.

D-Paste prepared at the time of use At the time of use, 80 g of the enzyme-polymer obtained according to Example 1, at 1200 units/g, and 20 g of glycerol are mixed until a thick paste is obtained.

When the paste has been obtained, it is applied to the surface of the wound in a sufficient quantity to cover it, and a compress is then applied in order to hold the dressing in place.

Claims (14)

1. A polymeric material capable of enzymatic action, which comprises a hydrophilic water swel!able carrier of crosslinked poly-ss-alanine carrying aldehyde groups, to which is covalent bonded at least one proteolytic enzyme.

2 A material according to Claim 1, in which the enzyme is a-chymotrypsin, trypsin, papain, fibrinolysin, streptokinase, streptodornase (Streptococcus haemolyticus) or bacterial (Bacillus subtilis) or fungal (Aspergillus orizae) proteolytic extract.

3. A material according to Claim 1, in which the proteolytic enzyme is collagenase.

4. A material according to any one of Claims 1 to 3 substantially as described in any of Examples 1 to 3.

5. A process for the preparation of the material according to any one of Claims 1 to 4, which comprises: 1) preparing a gel from spheres of crosslinked poly-ss-alanine in a buffer solution of pH from 6 to 9.5, 2) introducing the gel thus obtained into a buffer solution of the enzyme to be fixed, the said buffer solution being identical to that used for the preparation of the gel, 3) filtering off the gel carrying the enzyme and then washing with water to remove unbound enzyme.

6. A process according to Claim 5 in which step 1) is carried out at a pH of from 8.5 to 9.2.

7. A process according to Claim 5 or 6 in which the reaction with the enzyme is performed for 1 to 48 hours at approximately 4"C with agitation and under a nitrogen atmosphere.

8. A process according to any one of Claims 5, 6 or 7 substantially as described in any of Examples 1 to 3.

9. A material according to any one of Claims 1 to 5 produced by the process of any one of claims 5 to 8.

10. A medicament for cleansing wounds and assisting their cicatrisation, which comprises in an appropriate vehicle for local application, one or more polymeric materials as claimed in any one of Claims 1 to 5 or 9.

11. A medicament as claimed in Claim 9, in the form of a powder, the particle size of which is from 100 to 250 microns.

12. A medicament as claimed in Claim 10 which contains the polymeric material in the form of powder, in an excipient for ointment.

13. A cosmetic composition which comprises in a suitable vehicle one or more type of polymeric material as claimed in any one of claims 1 to 5 or 9.

14. A medicament or cosmetic composition as claimed in any one of Claims 10 to 13 substantially as described in Example 3.