FR2558171A1 - POLYMERIC MATERIAL WITH ENZYMATIC ACTION, PROCESS FOR PREPARING THE SAME AND USE THEREOF AS A MEDICAMENT - Google Patents

Abstract

ENZYMATIC ACTING POLYMERIC MATERIAL. THIS MATERIAL IS CHARACTERIZED BY THE FACT THAT IT CONSTITUTES A WATER-INFLATABLE HYDROPHILIC SUPPORT OF CROSS-LINKED POLY B-ALANINE CARRIING ALDEHYDES FUNCTIONS ON WHICH IS FIXED BY A COVALENT BOND AT LEAST ONE PROTEOLYTIC ENZYME. THIS MATERIAL IS APPLICABLE AS A MEDICINE FOR DETERSION AND AID IN WOUND HEALING.

Description

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  The subject of the present invention is a novel enzymatic polymer material, its method of preparation and its use, in particular as a medicament for the treatment of wounds with a view to their debridement and

of their healing.

  The debridement of wounds, burns, ulcers, etc. is commonly performed by enzymatic detergents which act on the exudates

and necrotic products.

  Among the commonly used enzymatic detersive agents, mention may be made of products based on trypsin, chymotrypsin or dna associated

  or not to other active ingredients.

  These products allow a good de-ionization, activation factor of

healing.

  Nevertheless, these can provoke phenomena of intolerance

  mainly due to an allergy to the enzymatic proteins used.

  Furthermore, the enzymes usually used in these detergent products lack heat stability, particularly at the use temperatures, where there is a significant loss of activity not only

  storage but also when the products are applied to the skin.

  It has now been found that the disadvantages of these detersive agents by enzymotherapy can be largely eliminated by fixing

  enzymes on a polymer support by covalent bonding.

  Indeed, the enzyme attached to the polymer is little or not resorbed at the wound which allows to reduce or eliminate, the

allergic phenomena.

  In addition, it was noted that the enzymes thus fixed had a good stability of activity substantially greater than the free enzymes to

  a temperature between 35 and 50 C.

  The improvement of the stability of the enzymatic activity over time makes it possible to significantly lower the quantity of enzymatic proteins.

  usually used in known detergent agents based on enzyme.

  The subject of the present invention is therefore, as a new industrial product, a polymeric material with enzymatic action consisting of a hydrophilic, water-swellable polymeric support of crosslinked poly-laylanine bearing aldehyde functional groups on which is fixed , by connection

  covalently, at least one proteolytic enzyme.

  The polymer support is obtained by crosslinking the polyalanine using a crosslinking agent, in particular using a dialdehyde, such as glutaraldehyde. Polyalanine is a known polymer whose preparation has

  has been described in US Pat. Nos. 2,749,333 and 4,082,730.

  The crosslinking process is generally carried out in suspension - 2 -

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  of the aqueous poly e-alanine solution in an organic solvent such as cyclohexane, toluene, methyl benzoate, benzyl benzoate, chlorobenzene or dichloroethane in the presence of a suspending agent such as

  cellulose derivatives, in particular ethylcellulose, ethyl-

  hydroxyethylcellulose and hydroxyethylcellulose, polyvinylacetate, malic anhydride-octadecylvinylether copolymer, maleic anhydride-octadecene copolymer, sorbitan oleates and condensation products of ethylene oxide and propylene oxide, known as the denominations

of "PLURONICS".

  The suspending agent as mentioned above has the essential object of orienting the formation of cross-linked poly e-alanine in the form of spheres, whose diameter is a function of the nature and proportion of

the suspending agent used.

  Thus, when polyvinyl acetate is used as the suspending agent, the cross-linked poly B-alanine spheres are generally of greater diameter than those obtained when using

  as a suspending agent for "Pluronic L84" or hydroxyethylcellulose.

  The proportion of the suspending agent also plays an important role in obtaining spheres of the desired diameter. It can vary in

  large proportions but is generally between 0.1 and 5%.

  The proportion of the crosslinking agent, that is to say dialdehyde, can vary in large proportions but is generally between 1 and 20% by weight relative to the weight of the poly-alanine

soluble in water reacted.

  The crosslinking reaction is carried out at a temperature of between 20 and 80 ° C. and at acidic pH, preferably between 1 and 2, obtained by

  addition of a mineral acid, for example hydrochloric acid.

  After stirring at room temperature, the reaction mixture is heated at a temperature between 40 and 100 C for a time

  Which may vary between 1 and 6 hours.

  The spheres obtained are then separated and then washed with the aid of an organic solvent or an alcohol / sodium hydroxide mixture and then with water and optionally

  ethanol and finally dried in an oven.

  Depending on the nature and the quantity of the suspending agent used, as well as the operating conditions such as, for example, the stirring speed, the diameter of the spherules of cross-linked polyalanine can vary.

between 2 and 500 microns.

  The spheres obtained can then be subjected to a sieving operation in order to separate those whose diameter is smaller or larger than a certain size, for example not included in the range of 100 -3-

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at 200 microns.

  By the method as described above, the spheres obtained, depending on the degree of crosslinking, are likely to swell in water by 2 to 25 times their initial volume. Moreover, depending on the proportion of dialdehyde used relative to the starting poly-B-alanine, the spheres obtained may have a greater or lesser proportion of functions.

  free aldehydes not having taken part in the crosslinking reaction.

  These aldehyde functions, which must be reduced when it is desired to use cross-linked poly-B-alanine as such, as absorption-detersive agents, are, on the other hand, absolutely essential for

  the attachment of enzymes by covalent bonding.

  In fact, the enzymes which carry primary amine functions can react on the aldehyde functions of the support, according to a Schiff base reaction, thus fixing by covalent bond the

enzymes.

  Among the various enzymes that can be fixed by covalent bonding to the cross-linked poly-B-alanine support, mention may be made in particular of: - the low specificity (broad-spectrum) proteolytic enzymes such as α-chymotrypsin, trypsin, papain, fibrinolysin, streptokinase, streptodornase (streptococcus hemolyticus), bacterial proteolytic extracts (Bacillus subtilis) or fungal (Aspergillus orizae), or more specific proteolytic enzymes (with reduced spectrum of action) such as collagenase whose activity is limited to collagens

native and denatured.

  In the fixing reaction which will be described hereinafter, a large excess of the enzyme which one wishes to fix is preferably used so as to avoid the existence of free aldehyde functions which could have

  a harmful influence, in particular to cause irritation phenomena.

  The process for attaching enzymes by covalent bonds consists of placing the spherules of cross-linked poly 6-alanine in a buffer solution of pH between 6 and 9.5, preferably between pH 8.5 and 9.2, or at a pH appropriate to the nature of the enzyme. The spheres, in contact with the solution

  buffer, swell and suspend.

  The mixture is then stirred for about 1 to 24 hours in a device allowing the arrival and the elimination of the buffer solution with a

  constant flow. The gel obtained is then wrung.

  The enzyme is then dissolved at 4 ° C. in a buffer solution identical to that used for the swelling of the crosslinked poly-8-alanine.

  solution to which is added the previously obtained gel.

  It is stirred for about 1 to 48 hours, and preferably for 3 to

  hours, at about 4 C, with stirring and under a nitrogen atmosphere.

  The enzyme-supported gel is then filtered with sintered glass and resuspended in distilled water at approximately 4 ° C. and the mixture is filtered off again and the washing is repeated until the washing waters have been removed.

  more enzymatic activity detectable.

  The spheres obtained are then dried, preferably by

  lyophilization, then subjected to sieving.

  According to the invention, the contact time necessary for the attachment of the enzymatic proteins depends, at 4 C, partly on the nature of the

  protein, on the other hand the pH of the process.

  The polymeric material according to the invention is preferably, before its use as a medicament, sterilized by any conventional means

especially by irradiation.

  The subject of the present invention is also, as a medicament for the treatment of wounds, in particular for their debridement and cicatrization, the novel enzymatic polymeric material as defined above either alone or in the form of a mix with another

  polymeric material whose fixed enzyme is of different nature.

  The medicament according to the invention can be used as it is in the form of a powder, the particle sizes of which are between 100 and. 250 microns, applied directly to wounds after

  washed with water or saline.

  The medicament may also be in the form of an ointment containing, in a suitable pharmaceutical excipient, the spheres in a

  proportion corresponding to the enzymatic activity sought.

  In addition to the various advantages mentioned above, the drug according to the invention makes it possible to ensure the debridement not only enzymatically but also by absorption, which results from the very nature of the hydrophilic support which has the property. to absorb the fluids from the wounds thus facilitating the process of healing. The medicament according to the invention thus makes it possible to ensure the debridement and the cicatrization of the wounds by a double action, that is to say by

enzymotherapy and absorption.

  According to one particular embodiment, the medicinal product may also contain, in combination, an active ingredient such as an antibiotic such as neomycin, a local anesthetic such as lidocaine hydrochloride (INN) or butoform (INN), an antiseptic such as -5 -

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  trichloro-3,4,4 'carbanilide or triclocarban (INN), an antifungal agent such as

  miconazole (INN) or an anti-inflammatory drug such as hydrocortisone.

  The drug according to the invention is particularly recommended for the treatment not only chronic traumatic, atonic, ulcerated but also vascular ulcers, varicose ulcers, post-phlebic ulcers, arterial ulcers, in the treatment of burns, pressure ulcers and more generally with regard to all wounds that

  have delayed healing.

  Although it has been more particularly referred to the use of the enzymatic polymeric material as a medicament, it is self-evident that it can also be used as a medicament.

  an active ingredient in cosmetic or dermatological compositions.

  Several examples of preparations of the medicament according to the invention and several examples will now be given by way of illustration.

application.

  EXAMPLE 1 Fixation of the trysine pH 9.2 to the ratio of cross-linked alanine-alanine in the form of resins In a 2-liter reactor equipped with a nitrogen inlet, a condenser, a thermometer, 600 g of distilled cyclohexane and then 3 g of ethyl cellulose T 100, sold by the company HERCULES, are then introduced into a dropping funnel and propeller stirring.

under stirring and under nitrogen.

  After cooling to 25 ° C., the stirring speed is; set at 1300 rpm, and a nitrogenous solution of

  polyalanine and glutaraldehyde obtained by dissolving 100 g of polyol

  alanine in 70 cc of previously degassed water saturated with nitrogen, then 1 g

  of ascorbic acid and 30 g of a 25% aqueous solution of glutaral-

  distilled dehydrogen and concentrated hydrochloric acid (d = 1.18)

to bring the pH to 1.

  The adiction completed, the mixture is stirred for 10 minutes at room temperature, then heated at 50 C for 3:30. After this la-ion time, the

  meliange is wrung out of Buchner's funnel.

  The crosslinked poly-p-alanine spheres obtained are then soomized in the following series of washes:

  (i) suspended in 800 g of cyclohexane 15 m.

  at reflux of the solvent, then spinings (ii) resuspended in 800 g of pure ethanol and then wringing, (iii) resuspension in water (800 cc) and then wringing, (this operation is repeated 3 times until neutrality of the last aqueous phase), - 6 -

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  and (iv) resuspending in the pure ethanol and finally spinning.

  The wet spheres are then dried on trays coated with filter paper for 24 h in ambient air and 24 h in an oven at 40 C. The spheres are then sieved on two sieves of mesh 250 and 100 microns leading to the obtaining of 50 g of a yellowish white powder from the

desired size.

  Measurement of swelling: A 10 ml test tube is filled with 1 ml of dry spheres with a diameter between 100 and 250 microns. It is then added to 10 ml by addition of water; the spheres swell rapidly to 5.7 ml (in

the absence of any agitation).

  b) Preparation of the crosslinked poly-B-alanine support at pH 9.2 4 g of cross-linked poly B-alanine in the form of spheres obtained above under a) are placed in 80 ml of a sodium borate buffer solution ( 19.7 g of Na 2 B 4 O 7, 10 H 2 O in 1 liter of water). The spheres swell rapidly and the mixture is stirred for 1 h in a device allowing the arrival and removal of the buffer solution with a constant flow rate of 60 ml / h. Then the gel is wrung on sintered glass whose inner pressure

is 30mm Hg.

  c) Fixation of the enzyme (trypsin) 477 mg of trypsin PROLABO assayed at 37000 Units / g are dissolved at 4 ° C. in 48 ml of a buffered solution at pH 9.2 as used for the preparation of the support, then the cross-linked poly 8-alanine gel obtained above under b) is introduced therein. The mixture is stirred vigorously for 24 h at 4 ° C. under a nitrogen atmosphere. The trypsin-supporting gel is carried on sintered glass, drained, then resuspended in 200 ml of distilled water at 4 ° C. and finally filtered again. This operation is repeated until the washing water no longer has trypsic activity, which requires at least four successive washes. The polymer obtained is brought

dry by lyophilization.

  Assay The activity is determined according to the method described in Biochemica Merck

  p.48. The assay shows an activity of 1200 Units / g.

  EXAMPLE 2: Fixation of the trypsin at pH 8.5 a) Preparation of the polyalanine support at pH 8.5 4 g of cross-linked poly B-alanine in the form of spheres prepared according to Example 1 a) are placed in 80 ml of a buffer solution (1000 ml solution containing 0.025 M sodium borate (Na 2 B 0.77, 10 H 2 O) and 152 ml of 0.1 M HCl solution). The spheres swell rapidly and the mixture is stirred for 1 hour in a device allowing the arrival and -7-

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  removal of the buffer solution with a constant flow rate of 60 ml / h.

  Then the gel is sintered on sintered glass whose internal pressure is

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 buffer solution at pH 8.5, as used for

  preparation of the support, and then introduced therein the gel prepared above under a).

  The mixture is stirred vigorously for 6 hours at 40 ° C. under a nitrogen atmosphere. The trypsin-supporting gel is carried on sintered glass, drained, then resuspended in 200 ml of distilled water at 4 ° C. and finally filtered again. This operation is repeated until the waters of

  washing no longer exhibit detectable tryptic activity (at least 4 washes).

  The product obtained is brought to dryness by lyophilization.

  Assay The activity of the polymer-enzyme is assayed according to the method described

  in Biochemica Merck p. 48. The assay shows an activity of 1800 Units / g.

  EXAMPLE 3: Fixation of α-chymotrypsin at pH 8.5 a) Preparation of the poly 8-alanine support 4g of cross-linked poly e-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 sodium borate (Na 2 B 4 O 7, 10H 2 O) and 152 ml 0.1 M HCl solution). The spheres swell rapidly and the mixture is stirred for 1 hour in a device allowing the arrival and removal of the buffer solution with a constant flow rate of 60 ml / h. Then the gel is drained

  on sintered glass with an internal pressure of 30 mm Hg.

  b) Fixation of the enzyme (α-chymotrypsin) 500 mg of a-chymotrypsin SIGMA assayed at 52,500 Units / g are put at 4 ° C. in solution in 60 ml of the buffer solution at pH 8.5, as used for the preparation of the support, and then introduced therein 5 g of cross-linked poly 8-alanine in gel form as obtained above under a). The mixture is kept for 24 hours under gentle stirring at 4 ° C. After this time, the gel containing α-chymotrypsin is drained on sintered glass, resuspended at 4 ° C. in 400 ml of distilled water and then again wrung . This operation is repeated until the washing water no longer has detectable chymotrypsic activity. The polymer-enzyme thus obtained is

brought to dry by lyophilization.

dosage

  The activity of the polymer-enzyme 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.

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EXAMPLES OF APPLICATION

  A - Powder for external use Duster flask containing the polymer-enzyme obtained according to Example 1, at 1200 Units / g having a particle size of between 100 and 250 microns. After cleaning the wound with water spray or saline, a sufficient amount of the polymer-enzyme is applied to cover the wound surface and a compress is then placed to hold the dressing. By renewing the application once a day

  rapid debridement of the wound and accelerated healing

  without noticing irritation phenomena such as those encountered

  when only the trypsin is used as a detergent.

  B - Spray powder for external use The following ingredients are packaged in an aerosol spray: - Polymer-enzyme obtained according to Example 2, at 1800 units / g ... 9 g - Magnesium stearate ....... .......................

....... 0.5 g - Isopropyl myristate .................................. ..DTD:. 0.2 g Difluorodichloromethane ..................................... 54 g Dichloro-1,2 tetrafluoroethane .............................. 36 g Before applying the powder to a wound, it is first of all cleaned with saline and a sufficient amount of the powder is applied to cover the surface of the wound and DTD: then places a compress to maintain the dressing.

  C - Hydrophilic ointment - Polymer-enzyme obtained according to Example 3, at 1464 units / g ... 7 g - Polyethylene glycol 400 ...................... ............

  ... 60 g - Polyethylene glycol 35/50 ................................... 33 g This ointment is applied to a wound after cleaning with water or saline and then a compress is applied in order to maintain the dressing.

  The application of the ointment is repeated once a day and there is an acceleration of healing without encountering any irritation phenomena. D - Paste prepared extemporaneously At the time of use, 80 g of the polymer-enzyme obtained according to Example 1 are mixed until a thick paste is obtained at 1200 units / g and

g of glycerol.

  The paste once obtained is applied to the surface of the wound in an amount sufficient to cover it and then a

  compresses to maintain the bandage.

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Claims (8)

  1. enzymatic polymeric material, characterized in that it consists of a water-swellable hydrophilic support of crosslinked polyalanine bearing aldehyde functions to which is attached by covalent linkage at least one proteolytic enzyme. 2. Material according to claim 1, characterized in that the enzyme has a broad spectrum of action and is taken from the group consisting of a chymotrypsin, trypsin, papain, fibrinolysin, streptokinase, streptodornase (Streptococcus hemolyticus), bacterial (Bacillus subtilis) or fungal (Aspergillus orizae) proteolytic extracts. 3. Material according to claim 1, characterized in that   the proteolytic enzyme has a reduced action spectrum and is collagenase.   4. Process for the preparation of the material according to claims 1 to   3, characterized in that it comprises the steps of: 1) preparing a gel from cross-linked poly 8-alanine spheres in a pH buffer solution of between 6 and 9.5, preferably at pH 8, 5 or 9, 2, 2) to introduce at about 4 C, the gel thus obtained in a buffer solution of the enzyme to be fixed, said buffer solution being identical to that used for the preparation of the gel, 3) to wring the gel supporting Itenzyme then to carry out several washes with the water until the absence of activity enzymatic detectable.   5. Method according to claim 4, characterized in that the enzyme-fixing reaction is carried out for a time of about 1 to   48 hours at about 4 C under stirring and under a nitrogen atmosphere.   6. Drug for detersion and wound healing aid, characterized in that it consists of or contains in a vehicle suitable for topical application, the polymeric material according to   any one of claims 1 to 4 or obtained according to any one of claims 5 and 6.   7. Medicament according to claim 6, characterized in that qp'i] is in the form of a powder whose particle sizes are between 100 and 250 microns.   8. Medicament according to any one of claims 6 and 7,   characterized in that it contains in an ointment   polymeric material in powder form.