MXPA00009733A - Method for sterilising native collagen in liquid medium, resulting native collagen, compositions containing same and uses - Google Patents

Abstract

The invention concerns a method for preparing native collagen, comprising pretreatment of the collagen in a mixer with double transverse cutters equipped with a system controlling agitating and shearing velocity and a thermostat, and a subsequent step of sterilising the collagen in liquid medium. The invention also concerns a sterile collagen, in particular a collagen mostly of type I, in native state and in particular pharmaceutical and/or parapharmaceutical and /or medico-surgical and/or ophthalmologic an/or cosmetic compositions and applications thereof.

Description

METHOD FOR THE STERILIZATION OF A NATIVE COLLAGEN IN MEDIUM LIQUID, STERILE NATIVE COLLAGEN OBTAINED BY MEANS OF THE SAME, COMPOSITIONS THAT CONTAIN IT AND ITS USES Description of the invention The present invention relates to a method for preparing collagen, comprising a step of sterilizing said collagen, to the sterile collagen obtained in this way, and to the compositions and uses, in particular the pharmaceutical and / or parapharmaceutical compositions and / or medical- surgical and / or ophthalmological and / or cosmetic, and the uses thereof. Collagen and its uses have been generally described in reference works such as collagen, Vol. 1, 2, 3 Marcel e. Nimni , CRC Press Inc, 1988 and Methods in Enzymology, Vol, 144, 1987, and Vol. 82, 1982, Ed. Leon W. Gunningham, for example. It is well known that collagen is a molecule with a three-dimensional structure and that it is present in most human and animal tissues and is found, in particular, in the skin, tendons and placenta.

Ref: 123397 Its various known properties are, among others, the humidification, the blood coagulation and the bleeding of wounds. In order to preserve its hemostatic action, it is generally desirable that collagen retain its native form prior to its administration in the human body, as described, for example in US-A-515, 637. In addition, due to its uses, in particular to its medical and / or cosmetic uses, the collagen must be free of any contamination (viruses, bacteria, prions, etc.) Collagen sterilization has been examined in various ways in the prior art, which has been confronted, in a few years, with the new challenges (risks of transmission of AIDS, Creutzfeldt-Jakob disease, etc.), due to the natural origin of collagen. The techniques for sterilizing collagen in solid form comprise the use of ethylene oxide in particular according to FR-A-2, 393, 581 or irradiation (use of beta or gamma ionizing radiation) as described for example in EP -A-0, 224, 453. The latter, which describes the cosmetic and pharmaceutical uses of the collagen-based sponge type, recommends sterilization in dry form due to the inability, according to that document, to sterilize soluble natural collagen. incorporated in emulsions or in solutions. EP-A-0,664,132 describes an adhesive composition for surgical use, based on non-crosslinked collagen, modified by oxidative cleavage. A preferred embodiment of the composition described in that document consists in the preparation of a sterile powder of type I non-crosslinked collagen [lagoon] by oxidative cleavage, and in the dissolution of this powder in water sterilized by ultrafiltration, by heating at approximately 60 °. C with agitation. It is specified that, in view of the controlled heating that is carried out the collagen loses its helical structure and is transformed into: gelatin. Furthermore, it is explained that the adhesive compositions representing the prior art in that document are based on cross-linked collagen, are not easy to handle and impose problems of application and mechanical resistance. EP-A-0,667,352 describes an alkaline treatment used on collagen in solution for the purpose of eliminating possible prions. In order to avoid any risk of residual contamination by said prions, this document teaches, more specifically, a method that consists in solubilizing collagen extracted from tissue, either by enzymatic digestion or by alkaline cleavage of the covalent bonds that interconnect the chains of collagen, and then in removing the tissue waste by filtration and holding the collagen solubilized in this way to an alkaline treatment. FR-A-2, 586, 703 teaches the preparation of transparent and physiological gels or collagen solutions based on type IV collagen. However, it is described that the type I, II or III collagens being practically insoluble at neutral pH, only allow the production of. opaque suspensions, and that it is impossible to use it.; to prepare transparent and physiological gels without resorting to chemical modification of the collagen molecules, which is likely to make them antigenic and / or toxic. Knowledge of this prior art research work has made it possible for the present inventor to demonstrate the following difficulties and problems that remain to date in the preparation of sterile native collagen, in particular type I.

* Mane] o The high viscosity of the native collagen, which imposes technical constraints in carrying out the filtrations, particularly in direct filtration procedures of conventional native collagen in liquid medium.

* Denaturation Partial or total denaturation by heat of the native collagen during the various usual sterilization treatments, which causes the loss of all or part of its structure and / or of its beneficial properties such as resorption, mechanical resistance or wetting, for example. - Sterilization by irradiation (ionizing radiation) or with ethylene oxide, which causes the deterioration of the advantageous active ingredients to be combined in solution with the collagen, such as antibiotics, for example and / or which causes a conventional gel based on collagen native is separated into two phases, making it unsuitable for commercial use. - Denaturation by sustained alkaline treatment.

* Toxicity and / or contamination (viruses, prions).

- Presence of traces of residual ethylene oxide in the products sterilized by this means / presence of contaminants in the initial crude collagen.

* Incompatibility Incompatibility of pH between the advantageous active principles that are soluble at neutral pH, such as anti-infective agents, for example, and collagen that is soluble in acidic medium.

* Galenic Formulation Unsatisfactory presentation of: preparations based on native collagen, which have a fibrous and heterogeneous appearance. Presentation of conventional preparations of biological adhesive, which is inappropriate given the use for which they are being routed (such as preparations that are not ready to be used, and which require a very long time of preparation, for example). Thus, the object of the present invention is to overcome the aforementioned difficulties and solve the aforementioned problems, in particular the sterilization of collagen, in liquid medium, while at the same time preserving its native form, and other problems that will be described later in the present. The object of the present invention is thus a method for preparing a collagen in native form, comprising a step of pretreating the collagen in a mixer with double transverse cutters, which is equipped with a system for controlling the speed of agitation and the cutting speed and with a thermostat, and a subsequent step of sterilizing the collagen in liquid medium. An objective of the present invention is also a sterile collagen, in particular a collagen mainly of type I, in a native form and compositions for human and / or veterinary use that contain it as the sole active ingredient, or in which this is combined with other advantageous substances that are acceptable from a pharmaceutical, parapharmaceutical, medical-surgical, and / or ophthalmological and / or cosmetic point of view. An object of the invention is also the use of the collagen according to the invention in the manufacture of a composition designed for the pharmaceutical and / or parapharmaceutical and / or medical-surgical and / or ophthalmological and / or cosmetic treatment of a human body or animal. These objectives and others will become apparent to those skilled in the art upon reading the following detailed description. The methods for preparing sterile collagen that are currently available impose the aforementioned implementation problems; previously and, to date, in practice, they have not allowed a direct sterilization of collagen in a liquid medium alone, or in the presence of other active ingredients, that is simple to carry out and that guarantees, in an effective manner, sterility and native nature of the collagen obtained, thus preserving its beneficial properties. Thus, the present invention has achieved this objective, and relates to a method for preparing collagen comprising a step, before sterilization, of mechanical pre-treatment, in a mixer, such as a "cutter mixer" sold under the name DITO-SAMA 175 (LA BOVIDA), and modified by J.F. I. Graulhet, France, for example, which is equipped with a temperature control system, of a non-sterile native collagen extract, which is solubilized and purified, optionally pepsinized. This pretreatment step allows, more surprisingly and unexpectedly, the sterilization in liquid form of the collagen and the preservation of its native form when it has not been pepsinized. Preferably, the collagen prepared in this way is a collagen mainly of type I.

The collagen mainly of the type] prepared in this way is sterile and in a native form, for example with secondary and tertiary structures, without change, and having retained all its beneficial properties, even after sterility. The collagen according to the present invention is useful in many applications that are detailed below, in particular: Veterinary human medical applications: pharmaceutical applications parapharmaceutical applications ophthalmic applications medical-surgical applications - cosmetic applications It can be used on its own, for example, without any other active principle, for example in the form of a clear sterile solution.

For injectable preparations or alternatively as a biological adhesive and in other forms that are detailed below. It can be used in compositions in combination at low pH, as a vehicle for other active principles for the immediate release of the latter. These other active ingredients are advantageously chosen from the group consisting of coagulation factors, for example thrombin, anti-infective agents, for example metronidazole, antibiotics, in particular macrolides such as gentamicin, fluoroquinolones such as pefloxacin, anti-inflammatory agents, examples are steroidal or non-steroidal acylcarboxylic anti-inflammatory agents or oxicam derivatives, antifungal agents and growth factors, in particular bone growth factors, such as somatotropin and epidermal growth factors such as EGF. However, in certain specific uses, for example in ophthalmology or in the case of medicinal combinations that incorporate active ingredients that are incompatible with an acidic pH, such as the glycopeptide antibiotic vancomycin, to mention only these two examples, it is obviously preferable to have a Collagen at neutral pH. It may also be advantageous to have collagen at neutral pH as a vehicle for a sustained release of active ingredients such as, for example, coagulation factors or alternatively, when a combination of the activity of the latter and a mechanical action is sought. Thus, it is also particularly advantageous to use the collagen according to the present invention. - or by combination, in solid stratified forms, of the two combinations prior to acid pH and neutral pH in specific applications that will be described later. As examples of the many presentations of the collagen according to the invention, mention may be made of: liquid forms: gel, in particular injectable gel, drops, foam, sprayed pseudosolid forms: films, sheets, membranes, bands or patches, etc., solid forms: powders, laminated / stratified sponges, films, membranes, threads, sutures, etc., It is also possible to exploit the properties; of acidity and solubility of the sterile collagen of: according to the invention in pharmaceutical or cosmetic compositions, in particular wetting compositions. It is possible, for example, to use the collagen at low pH and in pseudosolid form obtained according to the method of the invention, only c combined with other active ingredients such as vitamin E or vitamin C, in particular as a tissue humectant on the epidermis and as a vehicle for the active ingredient (s) in a face mask. These presentations and some others will be illustrated in the rest of the present description. In the context of the present invention: The term "native collagen" is intended to mean: a collagen that has not undergone modification in its initial structure, the integrity of its polypeptide chains (including telopeptides) and the helical structure of the molecule that has remained intact. The term "atelopeptide collagen" is intended to be understood: a collagen that has undergone limited proteolysis by enzymatic digestion using a proteolytic enzyme different from collagenase, such as, for example, trypsin, papain, etc., or better still pepsin, leading to the elimination of their telopeptides. Of course, if it were in native form, it retains its trihelicoidal structure. The term "sterile collagen" is intended to mean: a collagen that is sterile according to the standards of the World Health Organization, the European Pharmacopoeia and the United States Pharmacopeia (USP 22), for example, that have certified sterility after the microbiological analysis according to the criteria recognized by the related administrations.

The term "liquid collagen" is intended to mean: a collagen in solution in the form of a suspension, a dispersion or a gel. The term "pseudosolid collagen" is intended to mean: a collagen in the form of a gel that does not run and that forms a compact and homogeneous mass that contains more than 75% water. The term "solid collagen" is intended to be understood: a collagen containing no more than 10% to 25% water, and which may be in the solid form, for example in powder, film, sponge, membrane, etc. The phrase "native collagen with polymerizing power" is intended to be understood: a native collagen prepared according to a variant of the method according to the present invention, which has, in the liquid form or once solubilized from a solid form, a fluid nature at a temperature greater than 37 ° C. This is transformed into a homogeneous mass and is in a "pseudosolid" form at a temperature less than or equal to approximately 37 ° C. An atelopeptidic collagen may also have said polymerizing power. The phrase "solubilized and purified extract" is intended to mean: a non-sterile collagen extract at low pH (from 1.5 to 6.5, conventionally from 3 to 6, and even better from 4 to 5), in which the The content of sulfuric ash is generally less than 2% and the lipid content is generally less than 1% and comprises 12% to 13.9% hydroxyproline and 17% to 18.7% of the total nitrogen, and which does not contain traces. of tryptophan or polypeptide chains less than 95,000 Da. Such an extract can be obtained from dried dry limed skins, such as veal or rabbit skins, and / or connective tissues in particular from cattle, goats, pigs, sheep, horses, rabbits, deer, ostriches, fish, etc. ., or from dry or fresh bones of animals, corneas, or even tendons (e.g., ostrich Achilles tendons), or alternatively of purified collagen fibers or powders. This can also be extracted from placenta of human origin. The extract that is useful in the context of the present invention is prepared by a series of treatments comprising, in summary, the steps of: Liming / removing hair / washing / delimbing / removing the epidermis and subcutaneous tissues / cutting / Centrifugal drying / Sodium hydroxide immersion IN for 12 h at 25 ° C / wash to completely remove sodium hydroxide / grinding / film trimming / degreasing in a 0.01% Triton XlOO bath / optional inoglucan removal in a bath of potassium acid phosphate / washing / drying by centrifugation / washing with purified water / drying by centrifugation / acidification in acetic acid 10"2M, monochloroacetic acid or citric acid / optional dilution of the collagen extract The method of the present invention can be applied to various collagens of type I, II, III, IV, etc. However after this and purely by way of non-limiting illustration, the terms "collagen extract" and "collagen" refer, unless otherwise indicated, to a collagen or collagen extract primarily of type I. Thus, in the context of the present invention, the collagen extract consists mainly of collagen of the type I, for example, which contains more than 90% type I collagen. The extract may also consist entirely of type I collagen, for example by continuing the elimination of type III collagen according to the known techniques of dialysis, centrifugation and differential precipitation. with sodium chloride. Type I collagen is a high molecular weight polymer of 285,000 daltons consisting of the trihelical association of two identical polypeptide chains called cti (I) 2 and of a polypeptide chain called a2 (I) i. This belongs to the family of fibrillar collagens (type I, II, III and V). Collagen has a peak of denaturation that depends on its origin, the extraction method that leads to it, and its reticulated or non-reticulated form. In the non-crosslinked form, the denaturation peak is 35 to 45 ° C. The collagen mainly of type I has a ratio a2 (I)? / A2 (I) 2 of 0.48 to 0.52, as measured by densitometry carried out on polyacrylamide gel after migration of the chains by electrophoresis. Viral activation of this extract and the optionally present pepsin can be carried out by chemical treatment by passing alternatively through buffer solutions at pH greater than or equal to 13 and at pH less than or equal to 2.5. Treatments that are known per se are generally carried out, such as soaking in a bath of 4% calcium hydroxide, combined with 3% sodium sulphide, followed by a second bath of 0.5 sodium metabisulfite. % combined with 2% ammonium chloride. The optional neutralization of pepsin is carried out by sodium treatment which brings the pH back to 6.8-7.4, as described, for example, in FR 2,586,703. Similarly, the elimination of possible agents that are responsible for spongiform encephalopathies is bovine, and which are called "prions", it can also be carried out in a known manner, as described in WHO guidelines.

(WHO / COS / NHP / 92.104) and of the European Community dated December 11, 1991, for example.

This elimination can thus consist of a treatment with a sodium hydroxide solution IN for 1 hour at 48 hours at 25 ° C, or alternatively a treatment with sodium hypochlorite for at least 1 hour at 25 ° C. According to a first aspect, an object of the present invention is thus more precisely a method for the preparation of a collagen from a solubilized and purified extract, optionally pepsinized from native non-sterile collagen. Conventionally, the steps consist in mixing and cutting said extract in a mixer with double transversal cutters for a period of 1 minute to 60 minutes, advantageously 15 to 40 minutes, and preferably 10 to 20 minutes, at a stirring speed of 100. rpm at 10,000 rpm, generally from 500 rpm to 7000 rpm and preferably from 1000 rpm to 5000 rpm, while at the same time controlling the temperature, and then extracting said extract is sterilized. Advantageously, according to a first variant of implementation, and when the extract is not usually pepsinized (after this, the "variant 1" will be called, for simplicity, the modality of the method of the invention in which the extract is not pepsinized , except where indicated otherwise), the temperature, which is always * controlled, does not generally exceed 40 to 50 ° C, or even 60 to 80 ° C in the presence of a crosslinking agent (according to another embodiment) which is an alternative to this variant and is described later in detail).

Preferably, according to a first variant of the method of the present invention, since the extract is not pepsinized, its temperature increases gradually from room temperature until it reaches 40 ° -50 ° C in said mixer, while at the same time, the initial agitation speed is raised, in stages, from 500 rpm up to 5000 to 7000 rpm, and in each phase of the increase in viscosity, a dilution of the extract is carried out. The collagen is thus maintained at a chosen high viscosity, while at the same time its concentration is decreased, Advantageously, said high viscosity is from 15,000 to 20,000 cps (15 Pa.s to 20 Pa.s). For example, when said maximum temperature is 42 to 44 ° C, the extract is allowed to stand before the sterilization step. It is observed, after sterilization, that the collagen prepared in this way from a non-pepsinized extract has retained its native form and has acquired improved properties, particularly elasticity and mechanical strength. This has adhesive and polymerizing power. Preferably, said extract in the mixer is brought to room temperature at a temperature of at most 40 to 50 ° C, in steps of 2 ° C to 10 ° C, preferably 3 ° C to 5 ° C, while simultaneously increases the agitation speed, also in steps of 500 to 1000 rpm, to reach a speed of 1500 rpm at 7000 rpm, preferably 5000 rpm. It is in fact important to raise the temperature in stages since an abrupt increase in temperature temporarily denatures the collagen without sufficiently increasing its elastic properties. This temperature is brought to at least 40 ° C, since below this temperature the subsequent filtration becomes difficult or even impossible. It is also important to increase the speed of agitation in stages, to better control the high viscosity chosen, while at the same time decreasing the concentration of collagen as already mentioned. The concentration of collagen in the extract is from 0.001% to 15%, in general from 0.1 to 10%, and preferably from 3 to 6%. As described above, the temperature in the mixer can, however, reach higher values which can be as high as 80 ° C, in the presence of agents of . X crosslinking such as glutaraldehyde and azylic acids such as hydrazine, or any other crosslinking agent that is compatible with collagen. When these are present, these agents have a final concentration in the extract of 0.00075 to 0.1%, preferably 0.0075% to 0.01% in the case of glutaraldehyde, and from 0.1% to 2%, preferably 0.5% to 1.5%, for the hydrazine . The "cross-linked" collagen in this way consequently has a higher curing temperature which may be around 40 to 50 ° C, and has an in-resorption time which is longer compared to non-crosslinked collagen, and that can be as long as 8 weeks to 12 weeks. According to a particular embodiment of this variant of the method according to the invention, which consists in obtaining the sterile native collagen in the form of a viscous liquid, the following procedure is carried out: - A non-sterile native collagen, which is extracted from rabbit skins or ostrich Achilles tendons, is prepared at a sufficiently high concentration of approximately 10 to 15%.

- The non-sterile native collagen extract is then subjected to agitation and cutting in the mixer with double cross cutters already mentioned, at a moderate speed of 500 to 1000 rpm and at room temperature, of about 20 ° C. The centrifugation speed and the cutting speed are increased in stages, which has the effect of greatly increasing the viscosity of the extract. This viscosity can have the value of 7000 cps at 20,000 cps. From 1500 cps, the extract has a virtually elastic appearance, which makes it extremely difficult to shake and cut in the mixer. - In this phase of the increase in viscosity, a dilution is carried out in the extract by the addition of water for injection (WFI) or citric acid 10 ~ 2M, and the speed of agitation and the temperature in the mixer is increased in stages until a very high viscosity of about 20,000 cps (20 Pa.s) is obtained. - This operation is repeated several times, preferably four to five times until a temperature of 35 ° C, a speed of 3000 to 5000 rpm and a collagen concentration of about 3 to 6% is reached. The product is maintained at this temperature of 35 ° C and at this speed of 3000 to 5000 rpm for a few minutes, preferably 3 to 5 minutes. The temperature in the mixer is increased until it reaches 40 to 50 ° C, preferably 40 to 45 ° C, in the case of non-crosslinked collagen. At this temperature, the liquefaction of the extract is more surprisingly observed, with a drop in viscosity to about 40 to 100 cps. - The collagen extract, prepared as just described above, is immediately perfectly filtered, at a controlled operating temperature of 40 to 50 ° C, through membranes with a porosity of 0.45 μ to 0.22 μ. This then undergoes sterilization, preferably by absolute filtration through a membrane with a porosity of 0.22 μ, such as a Millidisck® membrane sold by the Millipore company, at the controlled operating temperature of 40 to 50 ° C. The filtration time at the temperature of 40 to 50 ° C, is chosen not to exceed 1 h, better still 10 to 20 minutes. It is also possible to carry out the sterilization, also preferably, by the addition of peracetic acid, produced in particular by the company Air Liquide under the name Soproper®, at the final concentration in the perfectly filtered extract of 0.5 to 50%, and preferably from 5% to 15%, by weight, with respect to the dry weight of the collagen. The neutralization of this sterilizing agent is carried out by the addition of sodium thiosulfate in a proportion of 2 to 20 g per 1 g of peracetic acid, and preferably from 4 g to 12 g / g of peracetic acid. The contact time required for sterilization by peracetic acid is from 1 h to 24 h at room temperature, preferably from 2 h to 4 h. According to another particular embodiment of this first variant of the method according to the present invention, which consists in obtaining the native collagen in the form of a pseudosolid gel, since the concentration of the collagen is from 0.5% to 10% is also advantageous sterilize it directly by irradiation at a dose of 2.5 Mrad (2.5 K 10"5 Gy) (ionizing radiation beta or gamma). It is surprisingly observed that the pseudo-solid gel remains compact and homogeneous and that, therefore, separation does not occur in two phases, contrary to conventional gels, and this retains its physico-chemical properties and its polymerizing power.This pseudosolid gel sterilized by this method can be advantageously liquefied to be combined with sterile active principles that are sensitive to ionizing radiation , such as coagulation factors, antibiotics, antifungal agents, etc., for the production of solid or liquid preparations uid, in particular the preparations cited as examples below: sponges, powders, films, membranes, patches, etc. When this method of sterilization is; used on solid preparations of collagen that are obtained according to this first variant: of the method, and that by themselves or combined with the. Active ingredients that are resistant to ionizing radiation and that are chosen from the therapeutic classes mentioned in the present document, there is no modification of the physicochemical properties or polymerizing and adhesive power of this. form of collagen.

According to this variant 1, the method according to the invention can also comprise a neutralization step. The neutralization can be carried out before or after the sterilization step. This neutralization can be carried out with sodium hydroxide, for example, in order to adjust the pH to 6.8-8.2, and preferably from 7.0 to 7.4. It is noted, more surprisingly, that this neutralization of the collagen in the viscous liquid and at a temperature of 40 to 50 ° C does not cause any precipitation in the collagen. The extract remains in clear translucent viscous liquid form, and quickly hardens under the same conditions as collagen at low pH. This is very advantageous, since the production of certain preparations such as one based on gel on collagen, or a combination of collagen with vancomycin, has been prevented to date due to the precipitation of collagen at neutral pH. The analysis of the collagen molecule of the invention by the conventional techniques already mentioned, confirms the integrity of the primary, secondary and tertiary structures of the collagen neutralized in this way.

The characteristics of the collagen obtained according to variant 1: It is noted, more surprisingly, that the collagen sterilized according to the first variant of the method of the present invention retains its native form. At a temperature of 40 to 50 ° C, this is a translucent and viscous liquid. Its viscosity varies in general from 20 cps to 300 cps (20 x 10"3 to 300 x 10 ~ 3 Pa.s), and preferably from 30 to 200 cps (30 x 10" 3 to 200 x "3 Pa.s), and still better from 40 to 100 cps (40 x ~ 3 to 100 x 103 Pa.s). Its pH is in general from 1.5 to 6.5, preferably from 2 to 5 and even better from 3 to 4. Its concentration is generally 0.001 to 15%, preferably from 0.1 to 10% and even better from 3 to 6%. It hardens rapidly at a temperature less than or equal to 38 ° C. It takes a pseudosolid form that has remarkable adhesive properties of hemostatic adhesive. Its hardening time at a temperature of about 38 ° C is less than 1 minute at a concentration greater than or equal to 5%, between 1 minute and 2 minutes at a concentration of 3 to 4%, and about 3 minutes at a concentration less than 2% The native sterile collagen obtained also has the other important characteristics or properties that follow: the collagen mainly, which contains for example 90% or more, of type I, for example a proportion a2 (I) i / di (I) 2 is 0.48 to 0.52 - water content: greater than 80% - appearance: liquid at a temperature greater than about 38 ° C, and sticky to the touch; compact and homogeneous gelled mass pseudosolide at a temperature of less than about 38 ° C. - total nitrogen: 17.0% to 18.7%. - Hydroxyproline: from 12% up to 13.9%. free of tryptophan, aminoglucans and molecular weight polypeptides < 95,000 daltons - lipids < 1% - sulfuric ash < 2% - denaturation peak: 38 ° C up 45 ° C, as measured by thermography (DSC). - adhesive power - polymerizing power - storage at room temperature - immediately ready for use.

Thus, an object of the present invention is also a collagen which has the aforementioned characteristics. Preparations based on the collagen obtained according to this first variant of the method according to the present invention and uses: As already mentioned, the collagen according to the present invention at a neutral acid pH has a polymerizing power and remarkable adhesive properties . The latter, combined with its hemostatic and resorption properties, gives it the quality of a resorbable hemostatic adhesive. The collagen of the invention can be preferably distributed in syringes or in bottles of 2 to 5 ml which are preferably made of glass and which are kept at a temperature that allows them to polymerize or harden between 0 ° C and 38 ° C, and even better at room temperature. Another advantageous form of this resorbable hemostatic adhesive according to the invention is the dry, powder or sponge form. This form does not require prior preparation before use. In order to obtain such a form, the collagen of the invention obtained in viscous form is subjected, for example, to lyophilization.

The collagen in liquid form of the invention is evenly distributed in containers (lyophilization trays), under aseptic conditions, leading to lyophilisates with a thickness of 1 to 20 mm, preferably 2 to 10 mm, and still better 3 to 5 mm. The obtained lyophilisate is cream colored and has a slightly elastic film-forming appearance. This has a water content of 1% to 25%, and preferably 5% to 15%. The lyophilizates can undergo grinding to give a powder of small, chosen particle size. For powder preparations, it is preferable to have a collagen concentration before freeze-drying of about 3 to 6%, or more, to ensure the best spraying or spraying. The powder after grinding can be distributed in sterile polyethylene bottles with a double lid, or another container that allows sterility to be maintained and to facilitate spraying on the hemorrhagic surface. In the case of sponges, the concentration of collagen should be relatively low, preferably 1 to 2%.

The film-forming sponges can be individually packaged, after lyophilization, in double bags or blister packs which are sealed by heat sealing. These are, for example, square, triangular or circular in shape and have a lateral length of a diameter of 1 cm to 30 cm depending on the uses. The action of the two dry forms; mentioned above is similar to that of the liquid form already described. However, the film-forming sponge also exerts a mechanical action that is often vital in the case of: hemorrhage and bleeding in backwaters, particularly when it is abdominal. This adhesive, whatever its form, in particular viscous liquid, pseudo-solid, film-forming sponge or powder, can be combined with active ingredients, in particular pharmaceutical active ingredients, such as for example coagulation factors which are involved in the acceleration of hemostasis (such as, for example, thrombin, fibrinogen, factor XII, etc.), or alternatively antibiotics in particular in the case of bone surgery.

When it contains coagulation factors, this adhesive can be advantageously combined with stabilizers, such as, for example, polyols (for example glycerol, or a polyalkylene glycol in which the alkylene radical has from 1 to 4 carbon atoms, such as polyethylene glycol or polypropylene glycol, in particular) or mono- or di- or polysaccharides (such as glucose or sucrose). As other preparations obtained from the viscous liquid form of collagen according to variant 1 of the method of the invention, mention may be made of: A pseudosolid patch, as a vehicle for active principles for local extraepidermal use, or alternatively a healing scar that is suitable in particular for patients with third degree burns, as well as a moisturizing mask for cosmetic use. The patch may incorporate one or more antibiotics, anti-inflammatory agents, antiseptics, antibacterial agents, antifungal agents and antifungal agents, and other physiologically and pharmaceutically active substances, in particular salicylic acid or nicotine. These active principles are added at a temperature higher than 40 ° C until homogenization is obtained. The mixture is degassed and propelled by a system under pressure, for example, such as a metering pump, and is distributed in a sealed container (ampoules or polyethylene films or the like, for example). After cooling the liquid to room temperature, the container is sealed by heat sealing. The collagen concentrations used in the patch are in the range of 1 to 8%, and preferably 2 to 5%. When these are present, the antibiotics have the following concentrations in mg of antibacterial / mg of collagen. Gentamicin: from 0.01 to 2 mg / mg, still better from 0.1 to 0.7 mg / mg and preferably from 0.15 mg / mg to 0.5 mg / mg. Vancomycin: from 0.01 to 2 mg / mg, still better from 0.1 to 0.7 mg / mg and preferably from 0.15 mg / mg to 0.5 mg / mg. Pefloxacin: from 0.01 to 2 mg / mg, still better from 0.05 to 0.5 mg / mg and preferably from 0.075 mg / mg to 0.25 mg / mg.

The thicknesses of the patch may be in the range of 1 5 mm or thicker. Patches of 1.5 to 2.5 mm thick however are preferred. The length and width are adjusted depending on the use considered. A patch obtained according to the present invention has a pseudosolid shape, is flexible, strong and highly hydrated (water content greater than 80%) and has a translucent appearance. This is easily applied to internal or external tissue surfaces, whether they are damaged or healthy. In the case of external epidermal uses, the patch can be fixed on an impermeable hypoallergenic adhesive that allows it to adhere to and be maintained on the surface of the tissue. The mask is obtained by emptying the collagen, on its own or combined with active ingredients such as, for example, vitamins (C, E, etc.), a surfactant such as benzalkonium chloride or humectants such as glycerol on a carrier belt. slow motion, for example, which makes it possible to obtain a homogeneous film of uniform thickness. After cooling, it is cut into suitable dimensions; in the form of pseudosolid, and it is packaged in airtight bags (made of aluminum with internal polyethylene lining, for example). The concentrations of collagen that are useful in the preparation of such a mask are generally from 0.2 to 3%, better still from 0.5 to 2% and preferably from 1 to 1.5%. The shapes and dimensions are by no means limited. A face mask conventionally has the shape and dimensions of a sheet of type A4 (21 x 29.7 cm). The thicknesses are generally 0.5 to 3 mm, and preferably 1 to 1.5 mm. The mask has a pseudosolid shape, is very flexible, soft to the touch and translucent, and is highly hydrated (water content greater than 97%). This is very easily applied to the skin. One or more colorants and other active ingredients that are compatible with collagen can be added to it. The spray contains the collagen and the active ingredients in the same concentrations and the same combinations as those described for the patch. This is advantageously packaged in _aa__as £ ás. ^ .__ afr_-. single dose flasks or sprayers of 5 to 25 ml, which are preferably made of glass or polyethylene. Before use, the collagen, which is in the polymerized form at room temperature, is liquefied by heating in a water bath or in an incubator for 2 to 3 minutes, in its original container. The collagen is sprayed onto the tissue surface from a distance immediately before it hardens. The collagen sprayed in this way adheres to the tissue surface and polymerizes rapidly on its surface, thereby forming a homogenous hemostatic / healing film. Such a spray is particularly suitable when the objective is a healing action and / or a sustained release of the active substances such as antibiotics, antiseptics, antifungal agents, anti-inflammatory agents, etc. This device for spraying from a distance has the advantage of avoiding any contact by the operator with the damaged tissue surface, and in this way any risk of possible local infection.

One of the uses of this spray, which is particularly advantageous and made possible due to the specific properties of the collagen according to the present invention, refers in particular to patients with third degree burns for whom a guarantee of sterility is a necessity. and of rapidity of care. Like other preparations obtained, from the viscous liquid form of the collagen, according to variant 1 of the method of the invention, it can be; Also make mention of: films or membranes: obtained when emptying on a polyethylene band, for example, followed by air drying, in a ventilated or vacuum incubator. The operating temperature is 10% at 35 ° C, and preferably from 20 ° C to 25 ° C. In order to obtain, for example, films with a thickness of less than 1 mm, preferably from 25 μm to 250 μm, collagen solutions are conventionally used in which the concentrations are in the range of 2 to 5%, preferably of 2.5 to 3.5%. The drying durations can be in the range of 1 hour to 120 hours. Advantageously, the drying durations are approximately 24 hours. Specifically, it is observed that the best results are obtained with relatively slow drying and a low air flow rate. The films or membranes obtained in this way have a water content of 1 to 15%, and preferably 5 to 10%. These are cut under aseptic conditions, either manually or automatically, and packed in sealed and waterproof containers. In a manner similar to the other forms mentioned above, the films or membranes may contain active ingredients such as antibiotics, antiseptics or also anti-inflammatory agents, etc. These can be produced from acidic (low pH) or neutral collagen. These are used for external or internal purposes for healing and / or release of an active principle, for example a medicinal one. Due to their elasticity on contact with a wet hemorrhagic surface, they can advantageously form a sheath that adheres to the area of the damaged tissue. In ophthalmological uses, these are produced from a collagen at a pH of 6.8 to 7.2, preferably. In this case, the films or membranes can take the form of a reservoir cylinder or transparent contact lenses, for a sustained local administration of the active ingredients to the eyes, for example. When you want to use this presentation of collagen in a liquid form (drops / eye wash), the collagen is preserved in capsules or other single dose containers, which must be reheated before use. In the case of ophthalmological uses, the collagen concentrations at neutral pH are, for the membranes or lenses, from 1% to 2.5%, and preferably 0.5% to 2%, and for the eye drops at neutral pH, the concentration of collagen it is from 0.1% to 1.5%, preferably from 0.2% to 0.5%. An object of the present invention is thus also the compositions described above for which, by way of illustration only, some of the possible pharmaceutical forms have been cited. According to a second variant of the method according to the present invention, the extract is subjected, before mechanical treatment in the mixer with temperature control, to limited proteolysis with a suitable enzyme, with the exception of collagenase, such as example trypsin, papain, or pepsin, preferably pepsin, as will be described later. In general, the concentration of collagen in the extract is from 0.1% to 2%, still better from 0.2% to 1%, and more preferably from 0.3% to 0.5%. Said pepsinized extract (purified, solubilized) is then treated as follows: Cascade clarification filtrations are carried out through membranes with various porosities, for example 100 to 50, and then 20 μm, 1 μm to 0.45 micrometers.

A new purification step is then carried out by differential precipitation with alkaline earth metal salts, for example with potassium or sodium chloride, preferably sodium chloride, optionally followed by dialysis against phosphate buffer of K2HP0 and Na2HP0, and finally, by centrifugation in order to recover the precipitated collagen.

The pellet obtained in this way is homogenized by gentle agitation in purified water, the collagen concentration being from 1% to 30%, and then washed 2 to 3 times before resuspending it in purified water and leaving it under stirring gently for 48 hours at a temperature of 0 ° C to 20 ° C, and preferably at a temperature of 0 ° C to 10 ° C.

This suspension is centrifuged or decanted with drying by centrifugation. The collagen is collected in a 10 ~ 2 M citric acid solution at a low pH of 2 to 5, preferably 2.5 to 3.5, the volume of which is calculated as a function of the final chosen concentration of the collagen.

The pellet is allowed to stand in this acid solution without any stirring at a low temperature of 0 ° C to 10 ° C for 2 hours to 48 hours, and preferably 12 hours to 24 hours. The collagen pellet swells, and loses its white coloration to become translucent.

The pellet is transferred to a mixer with double transverse cutters, such as, for example, the DITO-SAMA cutter / mixer already mentioned, and then the stirring and cutting are gradually increased, without exceeding 10,000 rpm, to achieve agitation, preferably from 1000 to 5000 rpm, and better still from 2000 to 3000 rpm, even more preferably 2500 rpm, while at the same time the increase in temperature is controlled in steps of 2 ° C to 10 ° C, preferably of 3 ° C to 5 ° C, not to exceed 35 ° C, preferably 25 ° C, until a total solubilization is obtained. The solution obtained is clear.

Again, the collagen is prepared from a collagen extract which is preferred and mainly of type I. According to this variant 2, the thus prepared and purified atelopeptide collagen solution can also advantageously undergo a sterilization by absolute filtration through a membrane with a porosity of 0.22 μm at the operating temperature, for example according to this variant, preferably at a temperature of 20 to 25 ° C, or alternatively by the addition of peracetic acid under conditions which are identical to those already described above, or alternatively by ionizing radiation (2.5 Mrads -2.5 x IO "5 Gy) in a pre-dried form which is optionally routed to be solubilized in sterile liquid medium in order to be combined with a sterile preparation of active ingredients that are sensitive to ionizing radiation and which are chosen from the therapeutic classes icas to which reference is made in this document. The optional neutralization is preferably carried out after the sterilization step at room temperature. At room temperature, it is observed that a precipitate begins to form from the second hour of the beginning of the neutralization. In this way, it is particularly advantageous to preserve the collagen at a temperature of: 0 ° C to 8 ° C, and to prepare the combinations or mixtures of the collagen with the other active ingredients, which are incompatible at low pH (from 1 to 6.5) , during the first two hours after the neutralization, preferably within the first hour, and even better within 30 minutes, and then start carrying out in particular the auxiliary operations of freezing, lyophilization or drying under air or vacuum .

Characteristics of the collagen obtained according to variant 2: The atelopeptide collagen prepared in this way is sterile and has retained its trihelical structure. This is in the form of a clear solution that has a low viscosity of 2 cps at 40 cps (2 x 10 ~ 3 to 40 x 10 ~ 3 Pa.s), and which is at acid or neutral pH, if required. The other characteristics of the collagen thus produced are as follows: collagen containing 90%, or more, collagen type I, for example a proportion a2 (I)? / Ot? (I) 2 from 0.48 to 0.52 - denaturation peak: 38 ° C to 45 ° C, as measured by differential thermography (DSC) total nitrogen: 17.0% to 18.7% hydroxyproline: from 12% to 13.9% tryptophan-free, aminoglycans and molecular weight polypeptides <95,000 lipid daltons < 1% sulfuric ash < 2% Thus, an objective of the present invention is also such sterile, atelopeptide native collagen.

Preparations based on collagen obtained according to variant 2, and their use. By way of examples, mention may be made of: - Use without modification in liquid form at acidic pH, such as a tissue or combination humidifier, in preparations with cosmetic active principles, for example, at concentrations of 0.1% to 3%; in combination in therapeutic wound preparations, for example in the presence of active ingredients in dermatology, such as anti-inflammatory agents, local antiseptics, anti-bacterial agents, anti-fungal agents, salicylic acid, etc. These formulations are in the form of cream, gel, foam or ointment for external use, with the collagen incorporated in them at concentrations of 1% to 3%. These can also be in the form of a liquid or a spray for external use, the collagen concentration being then from 0.1% to 1%. The active ingredients mentioned are used at known therapeutic doses.

Use at neutral pH in the form of paste or gel, in combination with phosphates and alkaline salts (sodium chloride, for example), then the concentration of collagen from 2% to 7%, for example, in the correction of depressions of the skin such as wrinkles or fine lines, or alternatively to strengthen the smooth muscles, such as in the case of urinary incontinence, or finally for the manufacture of tissue substitutes such as heart valves, for example, or bone substitutes, being then Collagen combined in particular with hydroxyapatite or with coral.

The use at neutral pH or acid in the preparation of a foam that contains a low concentration of collagen, and which is particularly useful in surgery and microsurgery for the preparation of damaged tissue and to ensure that the intertrust and parenchymal space is maintained, and avoiding anatomopathological adhesions between damaged or injured tissues (preferably in parenchymal, cardiovascular, thoracic, plastic or ENT surgery). This foam can incorporate active principles, in particular antibiotics, antiseptics and anti-inflammatory agents, in the same concentrations as those mentioned above for the preparations according to variant 1. Such foam will be described later; in more detail By using this sterile collagen according to variant 2, it is also possible to prepare, in particular with the aid of lyophilization drying, resorbable hemostatic sponges which are particularly advantageous due to their hemostatic action and to locally transport one or more active principles such as, in particular, anti-infective agents and coagulation factors. Such sponges obtained from the collagen at acid pH according to variant 2, are very advantageous, since they are instantly soluble once they have been put in contact with a liquid medium, which allows the immediate release of one or more active principles. The same sponges obtained from a collagen at neutral pH according to variant 2, the appearance of which is microfibrillar, allows sustained release and also exerts an advantageous mechanical action on the hemorrhagic surface to which they adhere. A collagen at acidic or neutral pH, at a concentration of 0.1% to 3%, preferably 0.2% to 2% and even better still from 0.5% to 1%, can be used in the preparation of such sponges. When these are present, the following antibiotics are used: gentamicin, pefloxacin and vancomycin, at the concentrations already mentioned for the preparations of variant 1. Other active ingredients may also be present, such as thrombin at a concentration, in International Units per mg. of collagen, from 0.1 IU / mg to 20 IU / mg, preferably 0.5 IU / mg to 10 IU / mg, and better still from 1 IU / mg to 5 IU / mg, or fibrinogen (in mg fibrinogen per mg ds collagen ) in a proportion of 0.1 mg / mg to 20 mg / mg, preferably from 0.1 mg / mg to 10 mg / mg and better still from 0.5 to 5 mg / mg. Other active agents can also be combined with this one, such as other blood derivatives, in particular prothrombin, factor Xa, factor IXa, factor XIII and any other activator or proactivator of the intrinsic and extrinsic coagulation mechanisms, optionally combined with other physiologically and pharmacologically active substances such as activators and inhibitors of the plasminogen, or anti-fibrinolytic agents, for example. Other substances can also be combined with these, for example specific enzymes that regulate the resorption of collagen, or alternatively cross-linking agents. The collagen mixtures according to the invention and the aforementioned active ingredient (s) are prepared in containers that are made of stainless steel, glass or polyethylene, and at temperatures of 5 ° C to 35 ° C, preferably of 10 ° C to 30 ° C, even better still from 15 ° C to 20 ° C. As already mentioned, when it is at neutral pH, the collagen must be used within the; 2 hours after its neutralization. The mixture is; then distributed in the freeze-drying containers that allow to obtain layers that are from 1 to 20 mm, preferably from 3 to 15 mm and better yet from 5 to 8 mm in thickness. These preparations can thus be lyophilized as a monolayer according to the following operating parameters: freezing temperature from -5 ° C to -60 ° C, absorption temperature from 20 ° C to 50 ° C, generally 25 ° C at 40 ° C and preferably from 30 ° C to 35 ° C. The preparations of sponges lyophilized in this way are white in color, and have a homogeneous, woven, velvety appearance. These have a water content of 1% to 25%. The sponges according to the invention can be used without modification, or they can undergo lamination in itself (in the lyophilizer chamber) by exerting an individual pressure on them or, on the outside under aseptic conditions, by passing through a roller mill. This operation makes it possible to increase the resistance of this type of sponge. Said preparations can be advantageously lyophilized in one or more layers. In this case, the adhesion between the layers occurs preferably during the freezing phase at chosen temperatures of -11 ° C to -20 ° C, preferably -11 ° C to -13 ° C. A laminate is then obtained in which the two lower layers, for example, can contain collagen at acidic pH combined for example with thrombin and fibrinogen (allowing the release of these products and the formation of a biological adhesive), and in which the The upper layer consists, for example, of collagen at neutral pH (making it possible to exert the mechanical action on the hemorrhagic surface as already mentioned). The aforementioned form makes it possible, very advantageously, to transport a biological adhesive without any prior preparation, in a manner contrary to known adhesives. Advantageously, the layer at neutral pH contains antibiotics, this allowing its sustained release, which is particularly advantageous in the case of bone infections. The choice of order and the number of layers is not important; this is within the reach of those skilled in the art who will adjust the latter depending on the uses considered. In these combinations according to the present invention, one may also include stabilizers, such as polyols, polyalkylene glycols or polysaccharides, or alternatively amino acids or other active agents that are comparable to collagen, such as anti-inflammatory agents, for example, or alternatively crosslinking agents as already mentioned, in order to increase, again advantageously, their resorption time. It is also possible to combine with these other resorbable agents which have a mechanical action and which make it possible to make sutures, such as cellulose or oxidized cellulose fabrics or Vicryl® (sold by Tenor company). In addition, the collagen obtained still according to variant 2 of the method of the invention, at acidic or neutral pH, possibly combined cor; Other active ingredients, can produce a hemostatic powder after lyophilization or drying. The collagen used then has a concentration of 1% to 3%. An object of the present invention is thus also the compositions described above, and for which some of the possible pharmaceutical preparations have been illustrated, which represent preparations of unpolymerized collagen obtained according to variant 2. After this, the non-limiting examples of some objects of the present invention will be given, and with reference to the drawings in which: - Figures 1 and 2 are diagrams of the polyacrylamide gel electrophoresis showing the separation of the polypeptide chains of collagen type I, respectively before and after the mechanical and thermal treatment of the invention (variant 1).

Figures 3 and 4 are diagrams of the heat denaturation of the collagen, demonstrating the native (non-denatured) nature of the collagen, before the mechanical and heat treatment of the invention (variant 1) and afterwards, respectively.

EXAMPLES EXAMPLE 1: Method for the preparation of a collagen with polymerizing powder (variant 1: non-pepsinized extract) The 10% collagen extract, prepared from ostrich Achilles tendons, as described above, is transferred to the mixer, with double cross cutters, already mentioned.

The stirring and cutting are then carried out at a speed of 1500 rpm and at room temperature (20 ° C) for 7 minutes. An increase in viscosity up to 15,000 cps is noted. The extract turns whitish. Successive dilutions are then carried out to reach a collagen concentration of 6%. After each dilution, the temperature is gradually raised by 4 ° C and the stirring speed is gradually raised by 500 rpm, at intervals of 3 to 4 minutes. When a stirring speed of 3500 rpm and 35 to 36 ° C is reached, these conditions are maintained for about 3 minutes, and then the stirring is stopped and the extract is allowed to stand at the same temperature of 35 to 36 ° C for 30 minutes. minutes A new dilution is then carried out (5 times) and the speed is increased to reach 5000 rpm, and the temperature is increased to reach 42 ° C, in 3 to 4 minutes. The collagen is then pasty and has a viscosity of 17,000 to 20,000 cps (17 to 23 Pa.s). The temperature increases to reach 44 ° C. Then, these conditions are maintained for 30 seconds to 1 minute; the pasty collagen liquefies quickly, becoming fluid in appearance, reducing its viscosity to 30-50 cps (30 x 10"3 Pa. s at 50 x 10" 3 Pa. s). The liquefied extract is immediately transferred to a filtration device under pressure that has been heat sterilized beforehand, and which is equipped with a double wrapping with water circulation that allows the temperature to be maintained at 42-45 ° C. The extract is then perfectly filtered through two. sterile filtration membranes, the first with a porosity of 0.45 μ and the second with a porosity of 0.22 μm. The filtrate is collected in a container under pressure, equipped with a double envelope, maintaining the temperature at 42-45 ° C. This filtrate is then subjected to absolute filtration through a membrane with a porosity of 0.22 μm. The collagen obtained is sterile, as confirmed by bacteriological tests according to the European Pharmacopoeia. In addition, analyzes are carried out before, during and after the mechanical heat treatments of the collagen thus obtained, which shows that the amino acid composition of the collagen remains unchanged. In addition, polypeptides of molecular weight less than 95,000 Da are not detected by polyacrylamide gel electrophoresis (Figures 1 and 2). It is also noted that the ratio a2 (I) ± / a ± (I) 2 is 0.49, for example a collagen containing more than 95% type I collagen. The thermographic analysis, by means of differential thermography (DSC) carried out with the help of the model DSC7 of the Delta Series of the company Perkin-Elmer and carried out during the heat and mechanical treatment of 40 to 50 ° C, confirms the disappearance of the peak denaturation of collagen, which corresponds to the loss of the trihelical structure of the molecule. However, this disappearance is only temporary, since the reappearance of the peak of denaturation is noted after the cooling of the extract of the collagen and its hardening (Figures 3 and 4). This is the proof that the loss of the trihelical structure of the collagen during the heat and mechanical treatment according to the present invention is reversible in nature. This group of results proves that the secondary and tertiary structures of the collagen prepared in this way are maintained. The other main characteristics and properties of the collagen obtained in this way are as follows: water content: 95% appearance: viscous liquid at a temperature greater than about 38 ° C, and sticky to the touch; pseudosolid gelled mass, compact and homogeneous at a temperature below about 38 ° C. viscosity: 45 cps (45 x 10 ~ 3 Pa.s) in liquid form - total nitrogen: 17.9% hydroxyproline: 12.8% free of tryptophan, aminoglycans and polypeptide chains < 95, 000 daltons pH of 5.5 - lipids < 1% sulfuric ash -2% The sterile collagen prepared as recent has been described is maintained at 42 to 45 ° C for a limited period of time in order to be rapidly distributed without modification in suitable containers or packaging such as in jars, syringes , blisters, etc., or after mixing with other active ingredients, such as coagulation factors, which have been sterilized beforehand, if required. When used alone, the collagen produced in this way forms an adhesive that has excellent hemostatic properties for general uses, such as parenchymal, cardiovascular, thoracic, orthopedic, ENT, urological, hepatic, visceral and gynecological-obstetric surgery, and in transplantation and bone marrow surgery; in microsurgery and any other medical field that requires the action of this medicinal form possibly combined with other active principles. This is maintained at room temperature, to which it hardens. This clearly distinguishes it from others, called biological adhesives currently available on the market, such as Tissucol® from the company Immuno-AG, Austria, or Biocolle® from Laboratories des Fractinnementes et des Biotechnologies, France, the dry form of which is necessarily stored at low temperature of approximately + 4 ° C. The collagen-based adhesive according to the present invention must be reconstituted in liquid form before being used. To do this, simply heating for 2 to 3 minutes is sufficient, for example in an incubator or in a water bath of 40 to 45 ° C, followed by slow manual agitation. Thus, in comparison with the above-mentioned known adhesives, the collagen-based adhesive according to the In addition to its advantageous properties, the invention has a preparation time that is approximately ten times shorter. When these are present, the coagulation factors are prepared beforehand, before being added to the collagen, in the following way. An amount of thrombin or fibrinogen powder corresponding to 2 IU / mg and 1 IU / mg of collagen, respectively, are collected in a 40 mM solution of calcium chloride (for example 1 ml of collagen for 50 IU of thrombin) and in water for injection (WFI). The filtration is then carried out separately through an absolute filtration membrane with a porosity of 0.2 μm. The thrombin or fibrinogen is then added to the sterile collagen which has been neutralized beforehand (pH 7.0) as already mentioned above. The mixture is homogenized and distributed. The distribution is carried out under pressure, for example using a dosing pump, in suitable containers, in particular glass jars or syringes, for example in the case of the preparation of biological adhesive; in heat-sealed blister-like bases or bags, as is the case, for example, with the patches. wetting films or masks, or alternatively in the trays of a lyophilizer in order to obtain a powder or a sponge, etc., as described above.

EXAMPLE 2: Liquid, sterile biological adhesive containing collagen + thrombin Using the product obtained in Example 1 in a sterile liquid form, a composition containing 2 ml of 5% collagen and an amount of thrombin corresponding to 200 IU is prepared in a glass jar. At the time of its use on a hemorrhagic surface, it hardens and adheres to the surface of the tissue, allowing closure of the lesion or incision, and acts as a hemostatic contact agent that causes the bleeding to stop.

EXAMPLE 3: Powder that are collagen + sterile fibrinogen Using the product obtained in Example 1, and after lyophilization and grinding of collagen combined with fibrinogen, in the form of a sterile powder, 360 mg of collagen containing 700 IU of fibrinogen per bottle are distributed in sterile polyethylene bottles. . This powder, when applied by spraying or spraying to a hemorrhagic surface, particularly in microsurgery in areas where access is difficult, makes it possible to stop bleeding through its hemostatic action on the intrinsic and extrinsic pathways of coagulation, and adheres to the hemorrhagic surface.

EXAMPLE 4: Sponge containing sterile collagen + thrombin Starting with 35 ml of sterile liquid collagen, which has a concentration of 1%, and which is obtained as described in Example 1 and then neutralized, it is distributed in trays or blister packs of 5 x 7 cm. A lyophilization is then carried out, and then the trays or the blister packs are closed under aseptic conditions, these trays or blister packs being then packaged in sterile double bags.

The sponge produced in this way makes it possible to cover an area of 35 cm 2, and corresponds to an amount of collagen of 350 mg that incorporates 700 IU of thrombin. This sponge has a fast hemostatic action that combines the action of collagen with that of thrombin on the mechanisms of coagulation, and adheres to the hemorrhagic surface allowing a considerable mechanical action to be exerted. Its film-forming appearance makes it possible to cover large hemorrhagic areas.

EXAMPLE 5: Patch containing collagen (pseudosolid) + metronidazole The anti-infective agent metronidazole, in liquid form and sterilized by pre-filtration, is added to the 2% collagen obtained in Example 1, in liquid form of 42 to 45 ° C and neutralized as mentioned above, in a proportion of 0.05 mg / mg of dry collagen. After cooling, the collagen that is in the form of a clear and homogeneous pseudosolid gel of 1 mm thickness is subjected to sterilization by irradiation with beta rays at 2.5 Mrad (25 x 10"5 Gy), Laboratoires Carie Orsay, France. It is surprisingly noted that the gel prepared from the collagen according to the present invention "does not break", for example it does not separate into two phases, in a manner contrary to a conventional gel that undergoes the same sterilization treatment by irradiation . This patch, when applied to an infected wound, has a topical and protective action, acting as a disinfectant due to the presence and release of metronidazole.

EXAMPLE 6: atelopeptide collagen with polymerizing powder (variant 1) Using a 10% extract of purified collagen that originates from the fresh ostrich Achilles tendon, the following procedure is carried out: the collagen is diluted by the addition of a 10"2 M citric acid solution at pH 2.5 until a collagen concentration of 1% is obtained, pepsin is added, 1/10 weight / weight of dry collagen, this is allowed to act at room temperature (20 ° C) for 7 hours, - on day 7, collagen it is neutralized with sodium hydroxide (30%) in order to obtain a pH of 7.2 Collagen is precipitated, non-collagen proteins and pepsin residues that remain in solution are eliminated by centrifugation with a centrifuge drying device / decanter such as, for example, that sold by the company Rousselet under the reference RC40, the pellet is washed in demineralised water, followed by drying by centrifugation, the weight of the water corresponding to times the weight of the pellet, and then a pellet of collagen in the form of wet fibers is obtained, this operation is repeated three times under the same conditions, collagen fibers being obtained in this way, with a water content of 70. %, the pellet is solubilized in citric acid 10"2 M at pH 2.5, the mixture is homogenized until the total solubilization occurs, and a collagen extract with a concentration of 10% is obtained, which is clear and has a viscosity of 6000 cps (6 Pa. S), the extract is transferred to the mixer with double cross cutters, already mentioned, and the procedure is continued according to the teaching of Example 1, until a collagen of about 5% is obtained. sterile atelopeptide native, with polymerizing powder, is particularly advantageous in ophthalmological uses, and may be in the form of a lens, flexible membranes or eye drops / washes, as previously described. s, the absence of potentially immunogenic telopéptidoe makes it especially attractive.

EXAMPLE 7: Collagen foam (variant 2) The foam is prepared from a pellet of moist collagen fibers, with a water content of 70%, which is treated according to variant 2 already described. This pellet is homogenized and maintained for 48 hours with gentle agitation at + 4 ° C in purified water. After centrifugation, the pellet is collected in a 10 ~ 2 M citric acid solution (pH 2.5), the collagen concentration being 2.5%. After standing for 24 hours at 4 ° C, the pellet is transferred to the mixer with double transverse cutters. At stirring speed and cutting speed and temperature, they increase gradually, and reach 2000 rpm and a temperature of 25 ° C. The extract is then filtered through an absolute membrane with a porosity of 0.22 μm at the operating temperature of 25 ° C. Benzalkonium chloride, at a concentration of 1.5 g / liter, is added to the sterile collagen obtained in this way, at 2.5%. The mixture is subjected to agitation progressing from 500 rpm to 2500 rpm under atmospheric conditions, the extract hardens and turns white, and thus the foam according to the present invention is obtained. The foam is distributed in dosing pumps of 10 ml or more optionally equipped with a catheter that allows its administration in areas where access is difficult, as is often the case in microsurgery.

In this way preserved, this foam is stable when stored at room temperature for at least 24 months. In the case of the presence of a heat-sensitive active principle, it is preferable to maintain the storage temperature for the foam produced in this way at 4-10 ° C. This foam is advantageously used particularly in parenchymal and thoracic surgery. This is particularly useful for maintaining inter-tissue and parenchymal spaces, and makes it possible to avoid anatomo-pathological adhesions between damaged tissues.

EXAMPLE 8: Stratified or monolayer hemostatic sponges (variant 2) The sponges are produced from the collagen obtained according to variant 2, as in Example 7, which is sterilized by absolute filtration through a membrane with a porosity of 0.22 μm. In order to obtain a monolayer sponge which also comprises a coagulation factor, for example, the present method is carried out: 35 ml of liquid collagen, at a concentration of 1%, which has been mixed in advance with sterile thrombin ( 700 IU) or with sterile fibrinogen (350 IU) and then neutralized, are distributed in ampoules of 5 x 7 cm. each of the aforementioned collagen combinations (the freezing and resorption temperatures are -25 ° C and 35 ° C respectively) are lyophilized conventionally and separately. The sponge containing collagen at neutral pH, obtained in this way, has a slightly fibrous, velvety appearance, and is respectively white and cream-colored in the presence of fibrinogen and thrombin. This sponge has a hemostatic action that is linked to the extrinsic and intrinsic action of collagen on the mechanisms of coagulation, which is further enhanced by the hemostatic action of the coagulation factors, namely thrombin and fibrinogen. In addition, the collagen at neutral pH as obtained according to variant 2, since it is insoluble, exerts a mechanical action on the surface of the hemorrhagic tissue which the coagulation factors can not exert when they are used alone, in the form lyophilized Furthermore, as already mentioned above, in such a presentation according to the present invention, the activity of the coagulation factors is fully preserved, in a manner contrary to conventional preparations (such as, for example, Tachocomb®, Hafslund Nycomed, described in EP-A-59 265), in which the latter are sterilized by ionizing radiation and are thus partially denatured. In order to obtain a stratified sponge, the following procedure is carried out: 18 ml of liquid collagen at pH 5, which is at a concentration of 0.5% and which has been mixed in advance with thrombin (700 IU) and with 1 ml of 40 mM calcium chloride, are distributed by 5 x 7 cm ampule, and then: gentle freezing is carried out at -10 ° C, and then 18 ml of 0.5% liquid collagen is added to this first layer at pH 5, which has been mixed in advance with fibrinogen (350 IU), and then the soft freezing is carried out again under the same conditions, a final layer of 1% liquid collagen, which this time is at a pH neutral, is added to the second layer obtained in this way, and finally lyophilization is carried out, as described above in the case of the monolayer sponge. In this way, a three-layered sponge is obtained which has the following remarkable properties: * once applied, the collagen at acidic pH is immediately solubilized, thereby releasing the coagulation factors that form a haemostatic adhesive on the surface hemorrhagic, * the outer layer at neutral pH also exerts a mechanical action, thus reinforcing the action of the hemostatic adhesive, everything else as if the hemorrhagic area were large. In addition, this sponge according to the invention has the advantage of not requiring preparation before use. This type of haemostatic presentation is particularly suitable in general in parenchymal, cardiovascular, thoracic, plastic, orthopedic, ENT, urological, hepatic, visceral and gynecological-obstetric surgery, bone marrow transplantation and surgery, and microsurgery, and in general in any field that requires such hemostatic action.

EXAMPLE 9: Sponge + vancomycin (variants 2) The following procedure is carried out: 6 liters of a 1.4% solution of collagen as obtained according to variant 2 of the method according to the present invention, they are introduced in a sterile container, and neutralized by the addition of sodium hydroxide, then 1 liter of a solution of vancomycin hydrochloride at 42 g / liter is added to the collagen solution which is maintained at room temperature, inside the hour after neutralization, then the solution is homogenized, and then, with the help of a dosing pump, the resulting mixture is transferred, through a sterile membrane with a porosity of 0.22 μm, to lyophilization vessels in a proportion of 70 ml per 10 cm container per side, and then, the freezing cycle is started to -25 ° C, followed by the sublimation and desorption cycle up to + 35 ° C, sterile sponges that are 7 mm thickness and containing collagen in a proportion of 8.4 mg of collagen / cm2 and 4.2 mg of vancomycin hydrochloride / cm2, or sponges that are 1 to 2 mm thick are finally obtained, exerting an automatic pressure on the surface of the layers inside the lyophilizer, in order to obtain a laminated sponge, and then the ampoules are sealed by heat and packed in double polyethylene bags, sterile. Identical sponges according to the invention are obtained by incorporating gentamicin, pefloxacin, or other antibiotics or anti-infective agents in place of vancomycin. These sponges, which locally and gradually release their active ingredients, are particularly useful in the treatment of many pathologies, in particular bone, visceral, traumatological and surgical pathologies, in particular in the prevention or treatment of possible microbial contamination. These sponges containing collagen according to the present invention, in addition to their excellent tolerance and capacity for biodegradation, have the great advantage of incorporating vancomycin which has been sterilized by filtration, as described above, and which has thus preserved all its activity, in a manner contrary to conventional preparations, in which this antibiotic is partially denatured by sterilization by ionizing radiation.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (13)

RE IVINDICATIONS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for preparing a collagen from a solubilized and purified, optionally pepsinized, extract of native non-sterile collagen, characterized in that the method comprises the steps of: i) shaking and shearing said extract in a mixer with double transverse cutters , while increasing the speed of agitation er steps, without exceeding 10, OOC rpm, and the temperature from 2 ° C to 10 ° C, preferably from 3 ° C to 5 ° C, to increase the final ambient temperature to a maximum temperature controlled, preferably less than 50 ° C, and then ii) sterilizing said extract in liquid medium, as a result of which a sterile collagen is obtained in a native or atelopeptide form.

2. The method according to claim 1, wherein the solubilized and purified extract is not pepsinized, the method characterized in that, in step (i), a dilution of the extract is carried out in each of the steps in which the viscosity of the The extract reaches a selected high value of 15 to 20 Pa.s, and, when the maximum temperature is 42 to 44 ° C, the extract is allowed to stand just before step (ii).

3. The method according to any of claims 1 and 2, characterized in that, before step (ii), the extract is perfectly filtered at the maximum temperature controlled through membranes with a porosity of 0.5 μm to 0.22 μm.

4. The method according to claim 1, wherein the solubilized and purified extract is pepsinized, the method being characterized in that the extract is treated as follows: - cascade clarification filtrations are carried out through membranes of various chosen porosities, up to 0.45 μm , and then the differential precipitation of the filtrate with sodium chloride is carried out, and because, in step (i), the maximum temperature reached is 35 ° C, preferably 25 ° C, and the stirring speed is 1000 at 5000 rpm, preferably 2500 rpm.

5. The method according to any of claims 3 and 4, characterized in that, in step (ii), an absolute filtration is carried out through a membrane with a porosity of 0.22 μm, or the extract is sterilized by addition of peracetic acid.

6. The method according to any of the preceding claims, characterized in that it comprises a neutralization step of the extract before or after step (ii).

7. The method according to any of the preceding claims, characterized in that the collagen is of the fibrillar type, preferably a collagen mainly of type I.

8. The method according to any of the preceding claims, characterized in that the collagen extract is obtained from dermal, connective tissues or animal tendons, in particular rabbit and ostrich Achilles tendons.

9. The native collagen or atelopeptidic type I, which can be obtained by a method according to any of claims 5 to 8, characterized in that it has the following characteristics or properties: a proportion a2 (I)? / Cc? (I) 2 from 0.48 to 0.52 sterile according to the standard of the European Pharmacopoeia - total nitrogen: 17.05 to 18.7% hydroxyproline: from 12% to 13.9% free of tryptophan, aminoglucans and molecular weight polypeptides < 95, 000 lipid daltons < 1% - sulfuric ash < 2%

10. A pharmaceutical and / or parapharmaceutical and / or medical-surgical and / or ophthalmological and / or cosmetic composition, characterized in that it comprises a collagen according to claim 9, alone or combined with at least one other active ingredient.

11. The composition according to the preceding claim, characterized in that the other active principle is chosen from the group comprising coagulation factors, for example thrombin, anti-infective agents, in particular metronidazole, antibiotics, in particular macrolides such as gentamicin, glycopeptides such as vancomycin, and fluoroquinolones such as pefloxacin, steroidal and nonsteroidal anti-inflammatory agents, acylcarboxyliae or oxicam derivatives, growth factors, in particular bone growth factors such as somatotropin, and epidermal growth factors such as EGF (epidermal growth factor), surfactants, humectants, stabilizers and vitamins.

12. A pharmaceutical presentation of a composition in accordance with any of lae; claims 10 and 11, characterized in that; selects from the group comprising gels, in particular injectable gels and pseudosolids gels, foams, eye washes, monolayer or stratified sponges, powders, plates or bands, masks, rubbers, films membranes, sheets and threads, in particular for sutures.

13. The use of a collagen according to claim 9, in the manufacture of a composition intended for the pharmaceutical and / or parapharmaceutical and / or medical-surgical and / or ophthalmological treatment of a human or animal body.