The method of sterilization of native collagen in a liquid medium obtained sterile native collagen containing compositions and their use

 

(57) Abstract:

The invention relates to an improved process for the preparation of collagen from solubilizing and purified, may pasensyahan extract non-sterile native or telopeptide collagen, including: i) the stage of mixing and shearing of the extract in mixer with dual lateral incisors with the gradual increase of the initial rate of mixing at 500-1000 rpm without exceeding the speed of 10000 rpm and the gradual increase of temperature for 2-10oWith, preferably 3-5oWith, thus, to increase the initial ambient temperature of the extract to the maximum controlled temperature, component not exceeding 50oWith, and then (ii) the stage of sterilization in the liquid environment of the extract with obtaining sterile collagen in native or telopeptides, native or telopeptide collagen type I, obtained with the above method, with the following characteristics or properties: value 2(I)1/1(I)2from 0.48 to 0.52; sterility in accordance with the standard of the European Pharmacopoeia; total nitrogen from 17,0 to 18.7%; hydroxyproline 12 is to the pharmaceutical and/or pharmaceutical, and/or medical-surgical, and/or vision, and/or cosmetic compositions. 4 C. and 9 C.p. f-crystals, 4 Il.

The invention relates to a method of producing collagen, including the state of the specified sterilization of collagen, thus obtained a sterile collagen, to compositions and use, in particular to pharmaceutical and/or pharmaceutical and/or medical-surgical, and/or vision, and/or cosmetic compositions and to the use of the latter.

Collagen and its application in General have been described in the papers cited as references, such as, for example, 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.

It is well known that collagen is a molecule with three-dimensional structure, which is present in most tissues of humans and animals and is found in particular in the skin, tendons and placenta.

Among his various known properties include, among others, the properties of hydration, blood clotting and wound healing.

To save the hemostatic action of collagen in General it is desirable to preserve its native form before introduction into the body, as indicated and/or in cosmetics, he must be free from any contaminants (viruses, bacteria, prions, and so on). Sterilization of collagen variously studied in the prior art, which, due to the natural origin of collagen, for several years, faced with new problems (risk of transmission of AIDS, the disease Creutzfeld - Jakob, and so on).

Methods of sterilization of collagen in solid form include the use of ethylene oxide in accordance, in particular from document FR-A-2393581, or radiation (beta and gamma ionizing radiation), as described, for example, in document EP-A-0224453. In the latter document, which describes cosmetic and pharmaceutical applications of the type sponge collagen-based, it is recommended sterilization in dry form due to the impossibility, in accordance with this document, sterilization soluble natural collagen included in the emulsions or solutions.

In the document EP-A-0664132 described adhesive composition for use in surgery, based on the collagen without the cross-linkage, modified by oxidative cleavage. One preferred embodiment of the compositions described in this document, is p the Kuna and the dissolution of the powder in sterile, subjected to ultrafiltration water by heating to approximately 60oWith under stirring. Stated that due to the ongoing controlled heating, collagen loses its helical structure and is transformed into gelatin. In addition, due to that adhesive compositions, representing the prior art in this document that are based on cross crosslinked collagen, easy to apply, and it is connected with problems of their application to the bonding surface and mechanical strength.

In the document EP-A-0667352 described alkaline processing used in relation to the collagen in solution, with the aim of eliminating possible prion. In order to avoid all risk of residual contamination indicated by prions, in this document, in particular, the method consisting in solubilization extracted from collagen tissue or through enzymatic processing, or using alkaline cleavage of covalent bonds, which provide the link between chains of collagen, and then removing the tissue fragments by filtration and the impact on the solubilized thus collagen alkaline processing.

In the document FR-A-2586703 is space specified because the collagens I, II or type III practically insoluble at neutral pH, they provide only an opaque suspension, and that it is impossible to apply them to obtain a clear and physiological gels without having to resort to chemical modification of collagen molecules that could probably do their antigenic and/or toxic.

Knowledge of prior art and research in the course of research work has allowed the author of the present invention to demonstrate the following difficulties and problems that still remain to date in obtaining sterile native collagen, particularly type I:

* Handling

- High viscosity native collagen, which contributes to technical limitations in the procedures of filtration, in particular in direct filtration conventional native collagen in a liquid medium.

* Denaturation

- Partial or complete thermal denaturation of native collagen during various commonly used methods of sterilizing treatment, which causes loss of all or part of its structure and/or its favorable properties, such as, for example, resorption, mechanical strength or At destruction favorable active components, to be combined in solution with collagen, such as, for example, antibiotics, and/or which leads to separation of the conventional gel-based native collagen, into two phases, making it unsuitable for commercial use.

- Denaturation under the influence of prolonged alkaline treatment.

* Toxicity and/or pollution (viruses, prions)

The presence of trace amount of residual ethylene oxide in the products sterilized with these tools, the presence of impurities in the raw source of the collagen.

* Incompatibility

- Incompatibility between pH favorable active constituent parts, which are soluble at neutral pH, such as, for example, anti-infective funds, and the pH of the collagen which is soluble in an acidic environment.

* Herbal composition

Poor form, which presents the products based on native collagen, which are fibrous and non-uniform appearance.

- The form in which presents the usual drugs of biological glue, unacceptable for the intended applications (e.g., such as drugs that are not ready for use and a cat who I is to overcome the above difficulties and resolving the aforementioned problems, in particular sterilization of collagen in a liquid medium, while at the same time its native form, and other issues that will be discussed below.

Thus, the object of the present invention is a method of producing collagen in native form, including the preprocessing phase of collagen in the mixer with dual lateral incisors, which controlled the speed of mixing and shear rate, and thermostat, and the subsequent stage sterilization of collagen in a liquid medium.

The object of the present invention is also sterile collagen, particularly collagen, predominantly type I, in native form and composition for use in humans and/or veterinary medicine, which contain as the sole active component or in which it is combined with other substances, which preferably is acceptable with pharmaceutical, para-pharmaceutical, medical-surgical and/or vision, and/or cosmetic point of view.

The subject of the invention is also the use of collagen in accordance with the invention in the manufacture of a composition intended for the pharmaceutical and/or parapharmaceutical or animal.

These and other objects will become apparent to specialists upon reading the following detailed description.

Existing methods of obtaining sterile collagen is created by the above-mentioned problems of implementation, and so far not possible in practice to carry out a direct sterilization in a liquid medium of the same collagen or collagen in the presence of other active substances. This sterilization is simple, and it is especially effective guarantees sterility and native nature of the obtained collagen, thus preserving its beneficial properties.

Thus, the present invention has achieved this goal relates to a method of producing collagen, including before sterilization stage mechanical pre-treatment in the mixer, such as a cutting mixer", commercially available under the trade name DITO-SAMA 175 (LA BOVIDA), and, for example, modified by "J. F. I., Graulhet, France", which is equipped with a system of temperature control of non-sterile extract of native collagen, which solubilizated and cleaned and, optionally, subjected to the action of pepsin. The most surprising and unexpected that this stage of preliminary education he was not subjected to pepsin.

Preferably thus obtained collagen is predominantly collagen type I.

Mainly collagen type I, thus obtained, is sterile and has negativnuu form, i.e., unmodified secondary and tertiary structures, and even after sterilization retains all its favorable properties.

Collagen in accordance with the present invention can be used in many fields of application, which are described in detail below, in particular:

various applications in medicine and veterinary medicine;

different types of pharmaceutical applications;

different types of pharmaceutical applications;

various applications in ophthalmology;

various types of medical and surgical use;

various kinds of cosmetic use.

It can be used by itself, i.e. without any other active constituents of substances, for example, in the form of a sterile transparent solution for injection drugs or alternative as a biological glue and other forms, which are described in detail below.

It can be used in compositions in combination with low pH as novtel is authorized parts are mostly selected from the group consisting of coagulation factors such as thrombin, anti-infective technologies, such as metronidazole, antibiotics, particularly macrolides, such as gentamicin, fluoroquinolones, such as pefloxacin, anti-inflammatory drugs, such as arylcarboxylic or aciclovir derivatives of steroidal or non-steroidal anti-inflammatory drugs, antifungal funds and growth factors, including growth factors bone, such as growth hormone, and epidermal growth factors such as EGF.

However, in certain specific applications, for example in ophthalmology or in the case of the use of drug combinations, including active components, which are incompatible with acidic pH, such as glycopeptides antibiotic vancomycin, it is shown in these two examples, it is obvious that it is preferable to have collagen at neutral pH.

It may also be preferable to have collagen at neutral pH as a carrier for the active constituent parts extended release, such as, for example, coagulation factors, or, alternatively, when it is envisaged the combination of recent activity and mechanical activity. So obrezinivanie previous two combinations in stratified solid forms at acidic pH and neutral pH for specific application methods, which will be described below.

As examples of many forms of collagen in accordance with the invention may be mentioned:

liquid form: gel, in particular, gel for injection, drops, foam, aerosol;

- pseudologia forms: films, sheets, membranes, tapes or pads, etc.;

- solid forms: powders, laminated/stratified sponges, films, membranes, sutures, suture material, etc.

You can also use the properties of acidity and solubility of sterile collagen in accordance with the invention in the pharmaceutical or cosmetic compositions, in particular in moisturizing compositions.

You can, for example, to use the collagen at low pH and in Pseudomonas form obtained in accordance with the method of the invention, alone or in combination with other active components, such as vitamin E or vitamin C, in particular, as the humidifier tissue on the epidermis and as a carrier for the active components (parts) in the face mask.

These and some other forms of presentation will be illustrated in the remainder of this description.

In the context of the present invention, the term "Nalini patterns, and retained the integrity of its polypeptide chains (including telopeptide) and the spiral structure of the molecule remains intact.

The term "telopeptide collagen" is intended to refer to collagen, which has been subjected to limited proteolysis using an enzymatic treatment using a proteolytic enzyme, other than collagenase, such as trypsin, papain, and so on, or better yet, pepsin, leading to the destruction of his telopeptides. Of course, if the collagen was in native form, it retains its trehserijnuju structure.

The term "sterile collagen" is intended to refer to collagen, which is sterile in accordance with the standards of the world Health Organization, the European Pharmacopoeia and United States Pharmacopeia (USP 22), i.e., which is certified as sterile after microbiological analysis in accordance with the criteria recognized by the administrations concerned.

The term "liquid collagen" is intended to refer to collagen in solution or in the form of a suspension, dispersion or gel.

The term "pseudology collagen" is intended to refer to collagen in gel form, which is not free-flowing and which adnanced to indicate collagen, which contains not more than 10 - 25% of water and which may be in solid form, for example in the form of a powder, film, sponge, diaphragm, etc.

The phrase "native collagen with the ability to polymerization" is intended to refer to native collagen, obtained in accordance with a variant of the method in accordance with the present invention, which is in liquid form or once solubilisation of the solid form has a liquid nature at temperatures higher than 37oC. He is transformed into a homogeneous mass, and is located in the "Pseudomonas" form at temperatures less 37oWith or approximates it. Telopeptide collagen may also have a specified ability to polymerization.

The phrase "the solubilized and purified extract" is intended to refer to non-sterile extract of collagen at low pH (from 1.5 to 6.5, usually from 3 to 6, better still from 4 to 5), in which the content of sulfur-containing ash is generally less than 2% and the lipid content of generally less than 1% and which contains from 12 to 13.9% hydroxyproline and from 17 to 18.7% of the total nitrogen, and which does not contain trace amounts of tryptophan or polypeptide chains less than 95000 Yes.

This extract can be polutnik tissues, in particular, from cattle, goats, pigs, sheep, horses, rabbits, young stallions, ostrich, fish and so on, or from dried or fresh bones, corneas, or even the tendons of animals (e.g., Achilles tendon ostriches), or alternatively from purified collagen fibers or powders. It can also be extracted from the placenta of human origin.

The extract, which can be used in the context of the present invention, is obtained through a series of treatments, including the end stages: processing lime / hair removal / washing / removal of lime / removal of the epidermis and subcutaneous tissues / cutting / centrifuge drying / dip 1 N. NaOH for 12 h at 25oWith / wash for the complete elimination of NaOH / grinding / cutting on film / degreasing in the bath Triton X100 at 0,01% / optional removal aminoglycan in the bath K2HPO4/ the wash / centrifuge drying / washing with purified water / centrifuge drying / acidification in 10-2M acetic acid, monochloracetic acid or citric acid / optional dilution of the extract of collagen.

The method of the present invention can be applied to various collagens type I, II, and "collagen" in the absence of other indications relate to extract collagen, predominantly type I.

Thus, in the context of the present invention the extract of collagen is composed primarily of collagen type I, i.e., containing more than 90% of type I collagen. The extract may also consist entirely of collagen type I, for example, with continued removal of collagen type III in accordance with known techniques, dialysis, centrifugation and differential precipitation with sodium chloride.

Collagen type I is a polymer with a high molecular weight component 285000 Yes, and consisting of the Association of the three spiral of two identical polypeptide chains, called 1(I)2and of polypeptide chains, called2(I)1. It belongs to the family of fibrillar collagens (type I, II, III and V).

Collagen has a peak denaturation, which depends on its origin, method of extraction, leading to him, and from his cross stitched or not cross stitched form. Do not cross stitched shape peak denaturation is when 35-45oC.

Collagen, predominantly type I has the value2(I)1/1(I)2from 0.48 to 0.52, as measured using densitometry carried out on polyacrylamide gel after migration CE is th pepsin may be performed using a chemical treatment by alternately passing through the buffer solutions at pH, greater than or equal to 13, and at pH below 2.5 or equivalent. In General, are such well-known by themselves ways of processing, such as soaking in a bath of calcium hydroxide at a concentration of 4% in combination with sodium sulfide at a concentration of 3% with the subsequent second bath of sodium metabisulfite at a concentration of 0.5% in combination with ammonium chloride at a concentration of 2%.

Optional neutralization of pepsin is carried out by processing sodium, which returns a pH level between 6.8 to 7.4, as described, for example, in document FR 2586703.

Similar known manner as described, for example, in the orders of the who (WHO/COS/VHP/92.104) and the European Community on December 11, 1991, can also be the elimination of possible agents, who are responsible for the bullish subcategorize encephalopathy and are called "prions". This elimination can thus consist of a processing solution of 1 n sodium hydroxide in a period of 1-48 h with 25oOr, alternatively, from the treatment with sodium hypochlorite for at least 1 h at 25oC.

In accordance with the first aspect, the object of the present invention is thus a more accurate method of obtaining collagen from solubilizing the stages consist of mixing and moving of the extract in mixer with dual lateral incisors during the period from 1 to 60 minutes, mostly from 15 to 40 min, and preferably from 10 to 20 min, with mixing speeds from 100 to 10,000 rpm, in General from 500 to 7000 rpm, and preferably from 1000 to 5000 rpm, with control at the same time, the temperature, and then in the sterilization of the extract.

Mainly in accordance with the first variant of realization, and when the extract is usually not ipsilaterally (for simplicity, hereinafter referred to as "option 1" - variant of the method of the invention, in which in the absence of other indications extract not pepinieres), the temperature is always controlled, in General, does not exceed 40-50oWith or even 60 - 80oWith the presence of cross linking agent (in accordance with another variant of realization, which is an alternative to this option and are described in detail below).

Preferably in accordance with the first variant of the method of the present invention, since the extract is not pepinieres, its temperature is gradually increased from room temperature up until the specified mixer will not be achieved temperature from 40 to 50oWith, at the same time gradually increasing the initial stirring speed of 500 rpm, in order to destinaton, supported by the high viscosity of the collagen, and at the same time decreases its concentration. Primarily indicated the high viscosity ranges from 15,000 to 20,000 CPS (15 to 20 Pas). For example, if the specified maximum temperature is 42 to 44oWith specified extract leave to stand before the specified phase sterilization.

After sterilization observe that thus obtained collagen nepersonifitsirovannaya extract retained its native form and acquired improved properties, in particular the elasticity and mechanical strength. It has a pin and polimerizuet ability.

Preferably, the temperature of the extract in the specified mixer speed is increased from room temperature to a temperature of a maximum of 40 to 50oWith 2-10oWith, preferably 3-5oWith, while also gradually increasing the stirring speed at 500-1000 rpm in order to reach speeds from 1500 to 7000 rpm, preferably 5000 rpm

Really important step temperature increase, since a sharp increase in temperature causes a temporary denaturation of collagen without sufficient increase ahoy subsequent filtering is difficult or even impossible.

It is also important step to increase the stirring speed in order to better control the selected high viscosity, at the same time reducing the concentration of collagen, as already mentioned.

The concentration of collagen in the extract is from 0.001 to 15%, generally from 0.1 to 10%, and preferably from 3 to 6%.

As described above, the temperature in the specified mixer may, however, reach higher values, which can reach 80oSince, in the presence of cross linking agents such as glutaraldehyde and Silovye acid, such as hydrazine, or any other cross linking agent, which is compatible with collagen. When the presence of these agents have a final concentration in the extract from 0,00075 to 0.1%, preferably from 0,0075 to 0.01% in the case of glutaraldehyde and 0.1 to 2%, preferably from 0.5 to 1.5% for hydrazine. "Cross stitched" thus the collagen has a higher temperature curing, which can be about 40-50oWith, and has time resorption in vivo, which is longer in comparison with collagen without the cross-linkage, and which can reach 8 to 12 weeks.

According to a special variant of realization of this variant of the method in bootverbose the following procedures:

Non - sterile native collagen, which is extracted from rabbit skins, or of Achilles tendon ostriches, get in high enough concentration of about 10-15%.

- Then extract non-sterile native collagen is subjected to agitation and shear in the above mentioned mixer with dual lateral incisors with moderate speed from 500 to 1000 rpm and at room temperature of about 20oC.

- The speed of rotation and the speed of the shift speed increase that takes effect in the form of a significant increase in the viscosity of the extract. The viscosity can have values from 7,000 to 20,000 CPS. Starting from 1500 CPS, the extract is essentially elastic look that makes it extremely difficult for its mixing and panning in the mixer.

At each phase, increasing the viscosity of the exercise dilution of the extract by adding water for injection (VDI) or 10-2M citric acid and stepped enhance the mixing speed and temperature in the mixer until then, until there is obtained a very high viscosity, amounting to about 20,000 CPS (20 Pas).

- This operation is repeated several times, preferably 4-5 times until, until you have reached temperature is the temperature of the 35oAnd this speed from 3000 to 5000 rpm for a few minutes, preferably from 3 to 5 minutes

- The temperature in the mixer increases until, until it reaches its level from 40 to 50oWith, preferably, in the case of collagen without the cross-linkage, from 40 to 45oC. At this temperature there is the most amazing dilution of the extract with the fall of the viscosity to approximately 40 to 100 SP.

- Extract collagen obtained by the above method, immediately carefully filtered through a membrane with a pore size of from 0.45 μm to 0.22 μm at a controlled operating temperature from 40 to 50oC.

Then it is subjected to sterilization preferably using absolute filtration through a membrane with pore size 0.22 μm, such as membrane Millidiscksold by Millipore, at a controlled operating temperature from 40 to 50oC. the filtering process at a temperature of from 40 to 50oWith chosen so that it does not exceed 1 h, even better that it was between 10 to 20 minutes Can also be sterilized, preferably also by adding peracetic acid produced, in particular, the company Air Liquide under matchtitle from 5 to 15 wt.% in relation to the dry weight of collagen. Neutralization of this sterilizing agent is carried out by adding sodium thiosulfate in the proportion of from 2 to 20 g per 1 g of peracetic acid, preferably from 4 to 12 g/g peracetic acid. The contact time required for sterilization of peracetic acid is from 1 h to 24 h at room temperature, preferably from 2 to 4 o'clock

In accordance with another specific embodiment of the implementation of this first variant of the method in accordance with the present invention, which consists in obtaining native collagen in the form psevdotegov gel, due to the fact that the concentration of collagen is from 0.5 to 10%, preferably to sterilize it right by irradiation in a dose of 2.5 Mrad (2,510-5Gray) (beta or gamma ionizing radiation). Unexpectedly observed that pseudology gel remains compact and homogeneous and that, in contrast to conventional gels, so there is no separation into two phases, and it maintains its physical and chemical properties and its polymerized ability. This pseudology gel, sterilized with this method, can mainly be liquefied in order to be combined with sterile active constituent parts, which are sensitive to Ionita liquid or solid preparations, in particular, drugs that are listed below as examples: sponges, powders, films, membranes, plates, etc.

When using this method of sterilization as applied to solid preparations of collagen, which is obtained in accordance with this first variant of the method, and which is used by itself or in combination with active components that are resistant to ionizing radiation and selected therapeutic classes listed in this document, no changes of physico-chemical properties or polymersomes or bonding ability of this form of collagen.

In accordance with this 1 option method in accordance with the invention may also include the step of neutralization. This neutralization can be performed before and during sterilization. The neutralization can be carried out, for example, sodium hydroxide to bring the pH in the range from 6.8 to 8.2, and preferably from 7.0 to 7.4. Most unexpectedly that, as noted, this neutralization of collagen in viscous liquid form and at a temperature of from 40 to 50oWith causes no deposition of collagen. The extract remains bright and transparent viscous liquid form and quickly hardens when those W is atstat production of certain drugs, such as gel-based collagen, or a combination of collagen with vancomycin, was the deposition of collagen at neutral pH.

Analysis of the collagen molecule of the invention with the aforementioned conventional techniques confirm the integrity of the primary, secondary and tertiary structures neutralized thus collagen.

Characteristics of collagen obtained in accordance with option 1.

The most unexpected was the fact that, as noted, collagen, sterilized in accordance with the first variant of the method of the present invention, retained its native form.

At a temperature of from 40 to 50oSince it is a viscous transparent liquid.

Viscosity generally ranging from 20 to 300 CPs (from 2010-3300 10-3Pas), and preferably from 30 to 200 SP (3010-3to 20010-3Pas), and even better from 40 to 100 CPs (from 4010-3to 10010-3Pass).

Its pH generally ranges from 1.5 to 6.5, preferably from 2 to 5 and even better still from 3 to 4.

Its concentration generally ranges from 0.001 to 15%, preferably from 0.1 to 10% and even better still from 3% to 6%.

It quickly hardens at a temperature below or equal to the 38oC. He adopts his hardening at a temperature of about 38oWith less than 1 min at a concentration of greater than or equal to 5%, from 1 to 2 min at a concentration of from 3 to 4% and about 3 min at a concentration below 2%.

Received native sterile collagen also has the following other important characteristics or properties:

- collagen is primarily collagen type I, i.e., it contains 90% or more, i.e. has the value2(I)1/1(I)2from 0.48 to 0.52;

- water content: 80%;

- appearance: liquid at a temperature higher than about 38oAnd sticky to the touch; Pseudotsuga dense and homogeneous jelly-like mass at a temperature lower than approximately 38oC;

- total nitrogen: 17,0 to 18.7%;

- hydroxyproline: from 12 to 13.9%;

- does not contain tryptophan, aminoglycan and polypeptides with a molecular weight <95000 Yes;

- lipids <1%;oSince, according to the measurement using a differential thermography (DSC);

- has the ability gluing;

- has the ability to cure;

- stored at room temperature;

- ready for immediate use.

So hebraistic.

The products based on collagen, obtained in accordance with this first variant of the method in accordance with the present invention, and methods of their use.

As already mentioned, the collagen in accordance with the invention has the ability to dry out and stick together at acidic or neutral pH. The last of these properties in combination with its hemostatic and resorption properties give it the quality rasskazyvaemoe hemostatic adhesive.

The collagen of the invention may preferably be distributed in the syringes or vials with a capacity from 2 to 5 ml, which is preferably made of glass and stored at a temperature that allows him to cure or harden at a temperature of from 0 to 38oWith even better at room temperature.

Another preferred form of this rasskazyvaemoe hemostatic adhesive in accordance with the invention is a dry, powder-like or spongy form. This form does not require any preparation before using.

To obtain this form of collagen of the invention, obtained in a viscous form, is subjected to, for example, lyophilization.

Collagen isawiyah, providing liofilizatow with a thickness of 1 to 20 mm, preferably from 2 to 10 mm and even better still from 3 to 5 mm, the resulting lyophilisate has a creamy color and has a slightly stretchy forming a film look. He has a water content of from 1 to 25%, and preferably from 5 to 15%. Lyophilizate can be subjected to grinding in order to obtain a powder with a selected small particle size.

For powder products preferably have before lyophilization concentration of collagen of approximately 3 to 6% or more, in order to provide the best atomization.

The powder after grinding can be distributed in sterile plastic vials with a double cap or another container, which provides the ability to maintain sterility and which facilitates the dispersion of bleeding on the surface.

In the case of sponges concentration of collagen should be relatively low, preferably from 1 to 2%.

Forming a film of a sponge can, after lyophilization be Packed separately in double bags or blister packs, which are closed thermal soldering. They have, for example, square, triangular or circular shape and, depending on with whom they form the same with the action already described liquid form. However, forming a film sponge also provides mechanical action, which is often vital in case of hemorrhage or bleeding in the cavity, in particular in the abdominal cavity.

Glue, regardless of its form, in particular in the form of a viscous liquid, psevdotegov substances, forming a film of a sponge or powder can be combined with active components, in particular pharmaceutical active components, such as, for example, coagulation factors, which are involved in the acceleration of hemostasis (such as, for example, thrombin, fibrinogen, factor XII, and so on) or, alternatively, antibiotics, in particular, in the case of surgical operations on the bones.

If the content of coagulation factors, the adhesive may preferably be combined with stabilizers, such as, for example, polyols (such as glycerol or polyalkyleneglycol in which alkalinity radical has from 1 to 4 carbon atoms, such as, in particular, polyethylene glycol or polypropylenglycol) or mono-, di - or polysaccharides such as glucose or sucrose).

Other drugs received from a viscous liquid form of collagen in accordance with option 1 of the method of the invention, it is possible the local application, or, alternatively, aerosol, contribute to scarring, which is suitable, in particular, for patients with third degree burns, and also as a moisturizing mask for cosmetic use.

The pad may include one or more antibiotics, anti-inflammatories, antiseptics, antibacterial agents, antifungal agents, and antimycotic agents and other physiologically and pharmaceutically active substances, in particular salicylic acid or nicotine. These active constituent parts are added at a temperature above 40oTo obtain homogenization. The mixture Tegaserod and operate with compressor device, such as a plunger pump, and distribute in hermetically sealed packaging (e.g. blister packs or plastic film or similar). After cooling liquid to room temperature, the package is closed by heat of soldering.

The concentration of collagen used in the overlay are in the range of from 1 to 8%, and preferably from 2 to 5%.

Antibiotics in case of their presence have the following concentrations in mg antibiotic 1 mg of collagen.

Gentamicin: from 0.01 to 2 is, even 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, even better from 0.05 to 0.5 mg/mg, and preferably from 0.075 mg/mg to 0.25 mg/mg.

Lining thickness may be in the range from 1 to 5 mm or thicker. However, the preferred lining thickness from 1.5 to 2.5 mm

The length and width are selected depending on the envisaged use.

The plate obtained in accordance with the present invention, has pseudowindow shape, is flexible, durable and highly hydrated (water content above 80%), and also has a transparent appearance.

It is easily applied on the inner or outer tissue surface regardless, damaged or healthy. In the cases of the outer epidermal application of the overlay may be placed on impermeable hypoallergenic adhesive that allows it to adhesion and retention on the surface of the fabric.

The mask obtained by pouring the collagen or in combination with active components, such as, for example, vitamins (C, E, and so on), surface-active substance, such as benzylaniline, or wetting agents, who ia a homogeneous film of uniform thickness. After cooling, cut it into pieces of suitable size in Pseudomonas form and packaged in sealed pouches (for example, made of aluminum with inner polyethylene layers).

The concentration of collagen that can be used in the preparation of this mask are in General from 0.2 to 3%, even better still from 0.5% to 2%, and preferably from 1 to 1.5%.

The shape and dimensions of the mask are in no way limited.

Facial mask for convenience has the shape and dimensions of the sheet type A4 (21 x 29,7 cm). The thickness generally ranges from 0.5 to 3 mm, and preferably from 1 to 1.5 mm.

The mask has pseudowindow shape, very flexible, soft, transparent and highly gidratirovana (water content of more than 97%). It is very easy to apply on the skin. It may be added one or more dyes and other active components, which are compatible with collagen.

The aerosol contains collagen and active ingredients in the same concentrations and the same combinations as those described for lining. He mostly Packed in bottles containing one dose, or aerosol cans with a capacity from 5 to 25 ml, which is preferably made of glass epared application thin with heat for 2-3 min in a water bath or incubator in its original packaging. Collagen directly in front of his hardening is sprayed in aerosol form on the tissue surface.

Sprayed thus aerosol collagen adheres to the surface of the fabric and very quickly polymerizes on the surface, thus forming a hemostatic and promotes scarring uniform film. Such aerosol, in particular, are suitable to ensure activities that contribute to scarring damage to the tissue, and/or prolonged release of active substances, such as antibiotics, antiseptics, antifungal remedies, anti-inflammatory agents, and so on).

This device for aerosol spray from a distance has the advantage that it allows to avoid any contact of the operator with the injured tissue surface, and therefore, any risk of local infection.

One of the applications of this spray, which has special advantages and the possibility of which is ensured thanks to the specific properties of collagen in accordance with the present invention applies, in particular, patients with burns of the third degree, which is necessary to guarantee sterility and speed of onset of therapeutic e the option 1 method of the invention, it is possible to mention: film or membrane, obtained, for example, by pouring onto a polyethylene tape with subsequent air drying in a ventilated incubator or in a vacuum. Operating temperature is from 10 to 35oWith, and preferably from 20 to 25oC.

To obtain, for example, films of thickness less than 1 mm, preferably from 25 to 250 microns, typically used solutions of collagen, in which its concentration is in the range from 2 to 5%, preferably from 2.5 to 3.5%. The drying duration can be in the range from 1 to 120 hours Mainly the drying duration is about 24 hours In particular observed that the best results are obtained

with relatively slow drying and relatively low speed of the air flow.

Thus obtained film or membranes have a water content of from 1 to 15%, and preferably from 5 to 10%. They are cut under aseptic conditions (either manually or automatically) and Packed in airtight and impermeable packages.

Similarly, like the above-mentioned forms, the film or membrane may contain active components, such as antibiotics, antiseptics or anti-inflammatory drugs, and so on, the OU ya external and internal purposes to accelerate healing and/or release of active components, for example, a medicinal product. Because of their elasticity when in contact with a bleeding damp surface they are mainly to form a membrane, which adheres to the area of damaged tissue.

When applications in ophthalmology, they are made of collagen at pH preferably from 6.8 to 7.2. In this case, the film or membrane may take the form of a cylindrical tank or transparent contact lenses for prolonged local delivery of active components, for example, in the eye. If you want to use these products of collagen in a liquid form (drops/liquid for rinsing eyes), retain collagen capsules or other packages containing one dose of that before use, must be hot.

In the case of use in ophthalmology concentration of collagen at neutral pH for membrane and lens is from 1 to 2.5% and preferably from 0.5 to 2%, and for eye drops at neutral pH, the concentration of collagen is from 0.1 to 1.5%, preferably from 0.2 to 0.5%.

Thus, the object of the present invention are also described above compositions, which are only to illustrate can be a PDF accordance with the present invention before and after machining in the specified mixer with temperature control, the extract is subjected to limited proteolysis with a suitable enzyme, except collagenase, such as, for example, trypsin, papain or pepsin, preferably pepsin as described below. In General, the concentration of collagen in the extract ranges from 0.1 to 2%, even better from 0.2 to 1%, and most preferably from 0.3 to 0.5%.

Then the specified ipsilaterally extract (purified and the solubilized) process as follows:

- Conduct brightening filtering through a series of membranes with different pore size, for example from 100 to 50 and then 20 μm, 1 μm to 0.45 μm.

Then conduct a new stage of purification by differential precipitation with salts of alkaline earth metals, such as potassium chloride or sodium, preferably sodium chloride, optionally followed by dialysis against phosphate buffer K2HPO4and Na2HPO4finally, using centrifugation for separation of the precipitated collagen.

Is thus obtained the ball homogenized with careful mixing in purified water, and the concentration of collagen is from 1 to 30%, and then washed 2-3 times before re-suspendirovanie in purified water and leaving for careful stirring for 48 hours at a temperature of from 0 or decant using centrifugal drying. Collagen is collected in a solution of 10-2M citric acid at low pH from 2 to 5, preferably from 2.5 to 3.5, the amount of which is calculated as a function of the selected final concentration of collagen.

- The ball leave this acidic solution without any mixing at low temperature from 0 to 10oWith in 2 to 48 hours, and preferably from 12 to 24 h Ball collagen swells and loses its white color, becoming transparent.

- The ball is transferred to the faucet with double cross cutters, such as, for example, the already mentioned cutter/mixer DITO-SAMA, and then the stirring speed and shift gradually increased, without exceeding the speed of 10000 rpm in order to achieve the stirring speed is preferably from 1000 to 5000 rpm, and even better from 2000 to 3000 rpm, even more preferably 2500 rpm, at the same time controlling stepwise increase in temperature from 2 to 10oWith, preferably, from 3 to 5oWith, in order to obtain solubilization not to exceed the temperature of the 35oWith, preferably 25oC. the resulting solution was transparent.

And in this case again the collagen is obtained from the extract of collagen, which preferably is essentially lopatenko collagen can then be mainly sterilization using absolute filtration through a membrane with pore size 0.22 μm at the operating temperature, that is, in accordance with this variant, preferably at a temperature of from 20 to 25oWith, or, alternatively, by adding peracetic acid under conditions that are identical to the conditions already described above, or, alternatively, using ionizing radiation (2.5 mrad) - 2,510-5Gray) on pre-dried form, which is not necessarily supposed to be solubilisate in sterile liquid medium for combining with sterile drug active components, which are sensitive to ionizing radiation and are selected from the classes of therapeutic agents, referenced in this document.

Optional neutralization is carried out at room temperature, preferably after step sterilization. It is observed that at room temperature the second hour neutralization and then begins to precipitate formed. Thus, it is of particular advantage is the storage of collagen at a temperature of from 0 to 8oWith the preparation of the combinations or mixtures of collagen with other active components that are incompatible with low pH (from 1 to 6.5) for the first two hours after neutralization, preferably within the first hour, and even proves the implementation or air drying or vacuum.

Characteristics of collagen obtained in accordance with option 2:

Thus obtained telopeptide collagen is sterile and retains its trehserijnuju structure. It is presented in the form of a transparent solution, which has a low viscosity of from 2 to 40 CPs (from 210-3to 4010-3Pas), and which, if required, has acidic or neutral pH.

Thus obtained collagen has the following other features:

- collagen, containing 90% or more of collagen type I, i.e. the ratio2(I)1/1(I)2from 0.48 to 0.52;

peak denaturation: from 38 to 45oSince, according to the differential thermography (DSC);

- total nitrogen: 17,0 to 18.7%;

- hydroxyproline: from 12 to 13.9%;

- does not contain tryptophan, aminoglycan and polypeptides with a molecular weight <95 000 Yes;

- lipids <1%;

The products based on collagen, obtained in accordance with option 2, and methods of their use. As examples can be mentioned:

- Application without modification in liquid form when titanime parts in a concentration of from 0.1 to 3%; in combination with accelerating scarring therapeutic drugs, for example in the presence of active components used in dermatology, such as anti-inflammatory agents, local antiseptics, antibacterial agents, antifungal agents, salicylic acid, etc., These compositions are presented in the form of a cream, gel, foam or ointment for external use, and collagen included in a concentration of from 1 to 3%. They may also be in the form of liquid or spray for external use, and the concentration of collagen is from 0.1 to 1%. Mentioned active constituent parts used in known therapeutic doses.

- Application at neutral pH in the form of a paste or gel in combination with phosphates and alkaline salts (e.g. sodium chloride), and the concentration of collagen in them ranges from 2 to 7%, for the correction of depressions in the skin, such as wrinkles or fine lines, or, alternatively, to strengthen smooth muscles, as in the case of incontinence of urine, or, finally, for the production of tissue substitutes, such as, for example, heart valves or Vice bones, and then collagen is combined, in particular, hydroxyapatite or coral.

- When the which can in particular, be used in surgery and microsurgery to repair damaged tissue and to preserve and interstitial lung space and avoid anatomo-pathological adhesions between injured or affected tissues (preferably, with operations in parenchymatous organs, cardiovascular, thoracic, plastic surgery or ENT surgery). This foam may include active components, in particular antibiotics, antiseptics and anti-inflammatories in the same concentrations as mentioned above for preparations as issued in accordance with option 1. This foam will be described in more detail below.

Using sterile collagen in accordance with option 2, you can also obtain, in particular, with drying by lyophilization, rassasyvanie hemostatic sponges, which have a special advantage because of their hemostatic action and because the local media of one or more active components, such as, in particular, anti-infective funds and/or coagulation factors.

Such sponges derived from collagen at acidic pH in accordance with option 2, have a great pre is Zelenogo release one or more active components, which is the active principle. The same sponge, obtained from collagen at neutral pH in accordance with option 2 and having a microfiber type, provide the possibility of long-term release of the current start and have a predominant mechanical action on the bleeding surface to which they adhere.

Collagen at acidic or neutral pH in a concentration of from 0.1 to 3%, preferably from 0.2 to 2% and even better still from 0.5 to 1%, can be used in the manufacture of such sponges.

For drugs of option 1 in case of presence of antibiotics, use the following antibiotic concentrations mentioned for products option 1: gentamicin, pefloxacin and vancomycin.

May also be present other operating beginnings, such as thrombin in a concentration in International Units per 1 mg of collagen from 0.1 to 20 IU/mg, preferably from 0.5 to 10 IU/mg, and even better from 1 to 5 IU/mg, or fibrinogen (mg fibrinogen per mg of collagen) in proportions of from 0.1 to 20 mg/mg, preferably from 0.1 to 10 mg/mg, and even better from 0.5 to 5 mg/mg. With them can also be combined with other active components, such as other blood derivatives in chastnih mechanisms of coagulation, optional in combination with other physiologically and pharmacologically active substances such as, for example, activators and inhibitors of plasminogen or antifibrinolytic funds. They can also be combined with other substances, for example, specific enzymes, which regulate the resorption of collagen, or, alternatively, the transverse cross-linking means.

A mixture of collagen in accordance with the invention and the above active constituent parts are prepared in containers, which are made of stainless steel, glass or polyethylene and at temperatures from 5 to 35oC, preferably from 10 to 30oAnd even better still from 15 to 20oC.

As already mentioned, at neutral pH the collagen should be used within 2 h after its neutralization. The mixture is then spread in lyophilization containers that provide the opportunity to obtain layers having a thickness of from 1 to 20 mm, preferably from 3 to 15 mm, and even better from 5 to 8 mm. These drugs can thus liofilizirovanny as a monolayer in accordance with the following functional parameters: temperature freezing -5 to -60oTemperatures resorption from 20 to 50oWith parathas sponges are white and have a uniform, wicker, "fluffy" look. The water content is from 1 to 25%.

Sponges in accordance with the invention can be used without modification or may be laminated in situ (in the camera lyophilizate) with exposure to or outside the individual pressure under aseptic conditions by means of passing through the rolls. This operation provides the ability to increase the strength of this type of sponge.

These drugs can be advantageously dried in one or more layers. In this case, the adhesion between the layers preferably occurs during the phase of freezing at selected temperatures ranging from -11 to -20oC, preferably from -11 to -13oC. Then receive a laminate in which two of the lower layer, for example, may contain collagen at acidic pH in combination with, for example, thrombin and fibrinogen (allowing release of these products and education biological glue), and in which the upper layer, for example, consists of collagen at neutral pH (providing the opportunity to have, as already mentioned, the mechanical action on the bleeding surface). The above form, in contrast to the known adhesive preparations, giving the network a great advantage. Layer at neutral pH mainly contains antibiotics that allows their continuous release, which has special advantages in the case of bone infections. The choice of the order and the number of layers is immaterial; it is subject to competence experts, who will select these indicators depending on the envisaged application.

In these combinations in accordance with the present invention may also include stabilizers, such as polyols, polyalkylene glycols or polysaccharides, or, alternatively, amino acids or other active tools that are compatible with collagen, such as, for example, anti-inflammatory drugs or, alternatively, as already mentioned, the cross linking agent for the newly predominant increase the time of their resorption. They can also combine other rassasyvanie tools that have a mechanical effect and which allow suturing, such as fabrics made of cellulose or oxidized cellulose, or vicryl(sold by "Ethnor").

In addition, the collagen at acidic or neutral pH, obtained in accordance with option 2 ways out is to provide for the possibility of obtaining hemostatic powder. Used then the collagen has a concentration of from 1 to 3%.

Thus, the object of the present invention are also described above compositions, for which you have illustrated some of the possible pharmaceutical forms, which are drugs unpolymerized collagen obtained in accordance with option 2.

Next, here will be given non-limiting examples of some of the objects of the present invention with reference to the drawings, in which:

- Fig. 1 and 2 are diagrams electrophoresis on polyacrylamide gel showing the separation of the polypeptide chains of type I collagen, respectively, before and after mechanical and thermal processing in accordance with the invention (variant 1);

- Fig.3 and 4 are diagrams of thermal denaturation of collagen, demonstrating native (not denatured) the nature of the collagen, respectively, before and after mechanical and thermal processing in accordance with the invention (variant 1).

EXAMPLES

Example 1. A method of producing collagen with polimerizuet capacity (option 1: nepaterizovanny extract).

Extract collagen at a concentration of 10%, obtained from filloval for 7 min conducting mixing and shift at the speed and frequency of 1500 rpm at room temperature (20oC). Increasing the viscosity of 15000 CP. Extract acquires a whitish color and has a supple appearance. This was followed by serial dilutions to get concentrations of collagen 6%. After each dilution, the temperature is gradually increased to 4oAnd the mixing speed is gradually increased to 500 rpm 3-4 minute intervals. On reaching the stirring speed is 3500 rpm and temperature 35-36oWith these conditions support for approximately 3 min, and then the stirring is stopped and the extract is left for 30 minutes to stand at the same temperature 35-36oC. Then spend new breeding (five times) and the speed increase to reach the level of 5000 rpm, and the temperature was raised to 3-4 min to reach the 42oC. the Collagen becomes paste-like appearance and has a viscosity 17000-20000 JV (17-20 Pas). The temperature increase in order to reach 44oC. these conditions are maintained for 30 sec - 1 min; pasty collagen quickly diluted, getting kind of liquid, and its viscosity is reduced to 30-50 JV (3010-3to 5010-3Pass).

Liquefied extract immediately transferred in a device for filtering under pressure, which pre the odes, allows you to maintain a temperature level from 42 to 45oC. Then, the extract was carefully filtered through two membrane for sterile filtration with a pore size of the first membrane of 0.45 μm, and the second is 0.22 μm. The filtrate is collected in a container under pressure, equipped with a double shell, and the temperature of the support at the level of 42 to 45oC. Then the filtrate is subjected to absolute filtration through a membrane with pore size of 0.22 μm.

The resulting sterile collagen, which is confirmed by bacteriological studies in accordance with the European Pharmacopoeia.

In addition, the analysis thus obtained collagen conducted before, during and after mechanical and thermal processing of collagen, show that the amino acid composition of collagen remains unchanged. Moreover, by electrophoresis on polyacrylamide gel not identify polypeptides with a molecular mass lower than 95000 Da (Fig.1 and 2). It is also noted that the ratio2(I)1/1(I)2is 0.49, i.e., collagen contains more than 95% of type I collagen.

Thermal analysis by differential thermography (DSC) carried out using the device model Delta-Series DSC 7 to zeznawanie peak denaturation of collagen, which corresponds to the loss trehspalnaya structure of the molecule. However, this disappearance is only temporary, because after cooling, the extract of collagen and its curing marked the re-emergence of the peak denaturation (Fig. 3 and 4). This is proof that the loss trehspalnaya of the collagen structure during thermal and mechanical processing in accordance with the present invention are reversible in nature.

This set of data proves that these are thus obtained secondary and tertiary structure of collagen.

Other key features and properties of the thus obtained collagen is presented below:

- water content: 95%;

- appearance: viscous liquid at a temperature higher than about 38oAnd sticky to the touch; Pseudotsuga compact and homogeneous jelly-like mass at a temperature lower than approximately 38oC;

- viscosity: 45 JV (4510-3Pas) in liquid form

- total nitrogen: 17.9 per cent;

- hydroxyproline:12,8%;

- does not contain tryptophan, aminoglycan and polypeptide chain with a molecular weight <95000 Yes;

- pH 5.5;

- lipids <1%;

When a stand-alone application thus obtained collagen forms an adhesive which has excellent hemostatic properties, making it suitable for use in General surgery, with operations in parenchymatous organs, cardio-vascular, thoracic, orthopedic surgery, otolaryngology, urology, liver, visceral and obstetric surgery and transplantation and surgery bone marrow; in microsurgery and any other medical areas that require action of this drug, perhaps in combination with other active components.

He was kept at ambient temperature at which it hardens.

This clearly distinguishes it from other currently available on the market so-called biological adhesives such as Tissucolfrom "Measurement AG, Austria, or Biocollefrom Laboratoires des Fractionnements et des Biotechnologies", France, the dry form which must be stored at low temperature is of expected to be recovered in liquid form. This is simple enough heat for 2-3 minutes, for example, in an incubator or water bath at 40-45oWith subsequent slow manual stirring.

Thus, in comparison with these known adhesives, adhesive collagen-based in accordance with the invention, in addition to its advantageous properties, has a cooking time of about 10 times shorter.

In the presence of clotting factors prepare them in advance, before they are added to the collagen, as follows.

In a solution of 40 mm of calcium chloride in water for injection (VDI) put the amount of powder thrombin or fibrinogen equivalent to 2 IU/mg and 1 IU/mg collagen (i.e. 1 ml of collagen by 50 IU of thrombin). Then separately are filtered through the membrane to the absolute filter having a pore size of 0.22 μm.

Then in sterile collagen, which beforehand was neutralized (pH 7.0), as already mentioned, add thrombin or fibrinogen. The mixture is homogenized and distributed in the tank.

Distribution in suitable containers made under pressure, using, for example, dosing pump, in particular in glass vials or syringes, for example, in SL is Oh, as in the case of, for example, plates, films or hydrating masks, or, alternatively, the trays lyophilizate to obtain powder or sponge and so on, as described above.

Example 2. Sterile liquid biological adhesive containing collagen + thrombin.

Using the product in sterile liquid form, obtained in example 1 in a glass vial composition is prepared containing 2 ml of 5% collagen and the amount of thrombin, corresponding to 200 ME.

During its application to a bleeding surface, it hardens and adheres to the tissue surface, allowing closure of injury or incision, and it acts as a contact hemostatic agent that stops bleeding.

Example 3. Powder containing sterile collagen + fibrinogen.

Using the product obtained in example 1, and after drying and grinding of collagen in combination with fibrinogen in sterile powder form, in a sterile plastic vials distributed to 360 mg of collagen containing 700 ME fibrinogen on the bottle.

The powder when applied by spraying on the bleeding surface, in particular, microsurgical operationalamplifier actions through internal and external mechanisms of coagulation, and adheres to the bleeding surface.

Example 4. Sponge containing sterile collagen + thrombin.

Starting with 35 ml of sterile liquid collagen, which has a concentration of 1%, which corresponds to the collagen obtained in example 1, followed by neutralization, its spread on the tray or blister packaging size 5 x 7 see Then spend the lyophilization and after that the tray or blister pack is closed under aseptic conditions, and these trays or blister packaging and then Packed in sterile double bags.

Thus obtained sponge provides the capability to cover an area of 35 cm2and corresponds to the number of collagen 350 mg, including 700 IU of thrombin.

This sponge has a rapid hemostatic effect, which combines the action of collagen with the action of thrombin in the clotting mechanisms, and adheres to the bleeding surface, providing significant mechanical impact. It forms a film type provides the possibility of covering large bleeding areas.

Example 5. Overlay containing collagen (pseudology) + metronidazole.

Anti-infective agent config in example 1, in liquid form at 42-45oWith and neutralized as described above, in a proportion of 0.05 mg/mg of dry collagen.

After cooling, the collagen, which is transparent and homogeneous psevdotegov gel 1 mm thick, is subjected to sterilization using irradiation with beta rays at 2,5 mrad (2510-5Gray), Laboratoires Caric Orsay, France.

Unexpectedly noted that the gel obtained from the collagen in accordance with the present invention is "not broken", i.e. not divided into two phases, in contrast to conventional gel, subjected to the same treatment sterilization by irradiation.

The gasket when applied to infected wounds has a local and a protective effect as a disinfectant due to the presence and release of metronidazole.

Example 6. Telopeptide collagen with the ability to polymerization (option 1).

Using a 10% extract of purified collagen derived from fresh Achilles tendon ostrich, conduct the following procedures:

- collagen is diluted by adding a solution of 10-2M citric acid at pH 2.5 to until will not be obtained, the concentration of collagen 1%,

type pepsin; 1/10 wt./wt. dry collagen,
- the ball is washed with demineralized water, followed centrifugal drying, the mass of water equivalent to 10 mass of the ball, and then get collagen bead in the form of wet fibers,

- this operation is repeated 3 times under the same conditions, and thus receive the collagen fibers with a water content of 70%,

- ball solubilizers 10-2M citric acid at pH of 2.5,

the mixture is homogenized until then, until complete solubilization, and the extract collagen with a concentration of 10%, which is transparent and has a viscosity of 6000 CPs (6 Pass),

the extract is transferred into the already mentioned faucet with double cross cutters and the procedure continues in accordance with the provisions of the example 1 to obtain about 5% of collagen.

This sterile telopeptides native collagen with the ability to polymerization has special advantages when used in ophthalmology and may be in the form of a lens, Flex monogenic of telopeptides makes it especially attractive.

Example 7. Collagen foam (option 2).

The foam produced from the bulb of the wet collagen fibers with a water content of 70%, which is treated in accordance with the already described in option 2. This bulb is homogenized and kept for 48 h with gentle stirring in purified water at +4oC. After centrifugation, the ball is placed in a solution of 10-2M citric acid (pH 2,5), the concentration of collagen of 2.5%. After settling for 24 hours at 4oWith the ball is transferred into a mixer with dual lateral incisors. The stirring speed, the frequency shift and the temperature gradually increased, reaching the level of 2000 rpm and a temperature of 25oC. Then, the extract is filtered through an absolute membrane with pore size 0.22 μm at the operating temperature of 25oC.

In thus obtained a sterile collagen in a concentration of 2.5% add benzylaniline at a concentration of 1.5 g/L. the Mixture is subjected to mixing under atmospheric conditions, increasing the speed from 500 to 2500 rpm, extract thickens and becomes white, and thus get the foam in accordance with the present invention.

The foam is distributed in dosing pumps with a capacity of 10 ml or more optional equipment is what happens in microsurgery.

Foam, canned so stable when stored at room temperature for at least 24 months. In the presence of heat-sensitive active constituent parts, it is preferable to maintain the temperature when storing the thus obtained foam level from 4 to 10oC.

This foam is mainly used, in particular, with operations in parenchymatous organs and structures of the chest. It can in particular be used for the maintenance and interstitial parenchymal spaces and provides the opportunity to avoid the formation of anatomical and pathological adhesions between injured or affected tissues.

Example 8. Stratified or single layer of hemostatic sponge (option 2).

The sponge is made from collagen obtained in accordance with option 2, as in example 1, which is sterilized with absolute filtration through a membrane with pore size of 0.22 μm.

To obtain a single-layer sponge, which also includes, for example, a coagulation factor, perform the following procedure:

- 35 ml liquid collagen at a concentration of 1%, which was pre-mixed with sterile thrombin (700 ME) or erased from the>/P>- each of the above combinations of collagen (at the temperature of freezing and resorption, respectively -25oWith the 35o(C) is subjected to regular and separate lyophilization.

Thus obtained sponge containing collagen at neutral pH has a "fluffy", slightly fibrous appearance, and in the presence of fibrinogen and thrombin has a white and cream color.

This sponge has a hemostatic effect, which is associated with the internal and external effects of collagen on the mechanisms of coagulation, which is additionally enhanced hemostatic action of coagulation factors, namely thrombin and fibrinogen.

In addition, the collagen at neutral pH, obtained in accordance with option 2, because of its insolubility, exerts a mechanical action on the bleeding surface of the fabric, which may not provide coagulation factors in dried form in a stand-alone application.

In addition, and as mentioned above, in this form of provision in accordance with this invention is fully preserved the activity of coagulation factors, in contrast to conventional forms of provision (such as Tachoco radiation and are therefore partially denaturirovannyj.

To obtain stratified sponge following procedures are used:

to 18 ml of liquid collagen at pH 5, which is contained in a concentration of 0.5%, which was pre-mixed with thrombin (700 ME) and 1 ml of 40 mm calcium chloride, spread on the blisters the size of 5 x 7 cm, and then

- conduct light freezing at -10oAnd then in this first layer, add 18 ml of 0.5% liquid collagen at pH 5, which was previously mixed with fibrinogen (350 ME), and then

again hold a slight freeze in the same conditions and in the second thus obtained layer add a final layer of 1% liquid collagen, which at this time has a neutral pH,

- conduct lyophilization, as described above in the case of single-layer sponge.

So, get a three-layer stratified sponge, which has the following distinctive properties:

- when applying collagen at acidic pH immediately solubilizated, releasing thus the clotting factors that are on the bleeding surfaces form a hemostatic adhesive

- the outer layer at neutral pH also exerts a mechanical action, thus enhancing the action of the hemostatic adhesive, someat the advantage of it does not require cooking before using.

This form of providing a hemostatic means particularly suitable for use in General, lung, cardiovascular, thoracic, plastic, orthopedic, otolaryngology, urology, liver, visceral and obstetric surgery during transplantation and surgery, bone marrow, microsurgery and generally in any area requiring such hemostatic action.

Example 9. Sponge + vancomycin (option 2).

Carry out the following procedure:

- 6 l a 1.4% solution of collagen obtained in accordance with option 2 of the method in accordance with the present invention placed in a sterile container and neutralized by adding sodium hydroxide, and then

in a solution of collagen, which has been stored at room temperature for 1 h after neutralization, add 1 l of a solution of vancomycin hydrochloride at a concentration of 42 g/l, then

the solution is homogenized, and then using the dosing pump the resulting mixture is transferred through the filter membrane with a pore size of 0.22 μm in the container for lyophilization in proportion sleduyushim cycle sublimation and desorption to +35oWITH,

- sterile sponges, which have a thickness of 7 mm and containing collagen in a ratio of 8.4 mg collagen/cm2and 4.2 mg of vancomycin hydrochloride/cm2or eventually with the help of automatic pressure at the surface layers inside lyophilizate to obtain a layered sponge receive sponges, which have a thickness of from 1 to 2 mm, and then

- conduct heat the solder to seal the blisters and the last is Packed in sterile plastic dual pouches.

Identical sponges in accordance with the invention are obtained from the inclusion of gentamicin, pefloksatsina or other antibiotic or anti-infective funds, instead of vancomycin.

These sponges, which are locally and gradually release their active components, can in particular be used in the treatment of many pathological conditions, in particular when it affects the bones, internal organs, in traumatology and surgical pathology, in particular for the prevention and possible treatment of microbial infections.

These sponges containing collagen in accordance with the present invention, in addition to their excellent tolerability and ability to undergo biological decomp what elytraria, as described above, and which, therefore, has kept all its activity, unlike conventional drugs, in which this antibiotic are denatured partially as a result of sterilization with ionizing radiation.

1. A method of producing collagen from solubilizing and purified, may pasensyahan extract non-sterile, native or telopeptide collagen, characterized in that it comprises the following stages: i) the stage of mixing and shearing of the extract in mixer with dual lateral incisors with the gradual increase of the initial rate of mixing at 500-1000 rpm without exceeding the speed of 10000 rpm and the gradual increase of temperature for 2-10oWith, preferably 3-5oThis is a way to increase the initial ambient temperature of the extract to the maximum controlled temperature, component not exceeding 50oWith, and then (ii) the stage of sterilization in the liquid environment of the extract with obtaining sterile collagen in native or telopeptides form.

2. The method according to p. 1, in which the solubilized and purified extract is not subjected to percenitaly, characterized in that in stage (i) carry out the dilution of the extract na JV (15-20 Pas), and when the maximum temperature reaches 42-44oS, and the viscosity will decrease the specified extract assert immediately prior to stage ii).

3. The method according to any of paragraphs.1 and 2, characterized in that prior to stage (ii) specified the extract carefully filtered with a specified controlled maximum temperature through membranes with a pore size of from 0.45 to 0.22 microns.

4. The method according to p. 1, and specified the solubilized and purified extract is subjected to percenitaly, characterized in that the extract is treated in the following way: spend cascade clarifying filtration through membranes with different selected pore size to 0.45 μm, and then spend the differential precipitation of the filtrate with sodium chloride, and in step (i) reach a maximum temperature of 35oWith, preferably 25oC, and the stirring speed is from 1000 to 5000 rpm, preferably 2500 rpm

5. The method according to any of the p. 3 or 4, characterized in that in stage (ii) provide absolute filtration of these extracts at the indicated maximum temperature through a membrane with pore size of 0.22 μm or sterilize the extract by adding peracetic acid at a concentration of 5-50% rasterization of the extract stage after sterilization ii) using peracetic acid, or after, or before stage sterilization ii) using membrane filtration.

7. The method according to any of the preceding paragraphs, characterized in that the collagen is a fibrillar collagen type, preferably collagen is predominantly collagen type 1.

8. The method according to any of the preceding paragraphs, characterized in that the extract of collagen derived from the skin, connective tissues and tendons of animals, in particular, from the skins of rabbits and Achilles tendons ostriches.

9. Native or telopeptide collagen type 1, which can be obtained using the method according to any of paragraphs.5-8, characterized in that it has the following characteristics or properties: value 2(I)1/1(I)2from 0.48 to 0.52; sterility in accordance with the standard of the European Pharmacopoeia; total nitrogen from 17,0 to 18.7%; hydroxyproline from 12 to 13.9%; does not contain tryptophan, aminoglycan and polypeptides mol.m. <95000 Yes; lipids <1%; sulfur-containing ash <2%.

12. Pharmaceutical form of the composition according to any one of paragraphs.10 and 11, characterized in that it is chosen from the group including gels, in particular, gels, injections and pseudologia gels, foams, liquids for rinsing eyes, single-layered or stratified sponges, powders, records or tapes, masks, sprays, films, membranes, sheets and filaments, in particular, for suture material.

13. Collagen under item 9 as an active ingredient of the composition, intended for the pharmaceutical and/or pharmaceutical and/or vision treatment of the human body

 

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The invention relates to medicine, namely to surgery, and may be applicable for dermatosurgical treatment gipotermiceski scars from surgical and/or therapeutic resurfacing

The invention relates to medicine, namely to abdominal surgery

The invention relates to medicine and concerns mediated virus amplified DNA transfer and method of improving the efficiency of transduction of hematopoietic and other cells under the action of retrovirus
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