The method of obtaining sterile collagen base "fibricor"

 

(57) Abstract:

The invention relates to medicine, in particular to methods of producing sterile collagen framework for the treatment of wounds, implantation. To the acetic acid solution deleteoperation collagen added under stirring methenamine or a solution of alkali metal salts mixture to pH of 4.3 and 5.3. Loose fibers are washed with water. Remove excess liquid phase to the protein content of 1.20 - 3.50 percent. Pasty substance stabilized with glutaraldehyde, treated with hydrogen peroxide solution, washed to remove excess liquid phase and sterilized by irradiation at doses of 15 to 20 kGy. The selection of the collagen fibrils can be produced in the presence of up to 10% of collagen glycosaminoglycan. In the composition of the base may include drugs, resistant to radiation, powder of hydroxyapatite or bone meal. The method allows to allocate fibrillar collagen deleteoperation collagen. 4 C.p. f-crystals, 6 other

The invention relates to medicine, in particular to methods of producing drugs for the treatment of wounds, implantation.

A method of obtaining ointments on the basis of collagen for the treatment of wounds [1]. In the high-viscosity solution cgenie them inside the body requires sterilization ointment. However, sterilization of the finished ointment and liquid collagen framework is very complex and still unresolved problem. A solution of collagen when heated above 37oC denaturised, the irradiation dose greater than 0.3 Mrad turns into a non-leaking gel. Methods for chemical sterilization of the finished ointment on collagen basis is unknown.

A method of obtaining suitable for injection fermentative collagen, mixed with a polymerization activator. The disadvantage of this method is the presence of 2 solutions - the solution of collagen and the activator solution, which are mixed at a low temperature before use. For sterilization of the solution of collagen using 1 M acetic acid, to reduce the acidity of which is then re-use dialysis against 0.001 M acid [2].

Closest to the claimed method is a method of obtaining injectable material consisting of cross stitched reconstructed particles of collagen fibrils [3]. The material has a high resistance and is suitable for introduction into the various cavities of the body with a syringe. But this method is time-consuming sterilization process the source of the collagen substrate associated with long-term di is h, forming fibrils at a neutral pH range of 6.0-8.0. Alkali-treated collagen under these conditions is stable and does not form fibrils. The methods used in patents [2,3] is not suitable for allocation fibrillar collagen from deleteoperation collagen.

In the present invention proposes a method of obtaining sterile collagen base "fibricor from acetic acid solution deleteoperation collagen, which under intensive stirring urotropine in the ratio of collagen-methenamine /2-10/:1 to a pH of 4.3-5.3 and washed precipitated fibrils, remove excess liquid phase to a protein concentration of 1.20-3.50% and pasty fibrillar basis Packed in plastic packaging, sterilized by irradiation with a dose of 20 to 25 kGy.

The essence of the invention lies in the fact that by mixing an acidic solution of collagen and urotropine the decomposition of the latter with ammonia and formaldehyde, which increases the pH to the isoelectric condition, the deposition of collagen fibrils with their simultaneous structuring present molecules of formaldehyde. The deposition of collagen fibrils occurs at the minimum ratio of collagen/methenamine 10 : 1. Polyatomic fibrils combination of collagen and urotropine is carried out at intensive mechanical stirring, for example in a homogenizer RT-1 at 4000 rpm

The resulting fibrils are insoluble in water, so you can completely remove the products of its decay. From the washed fibrous substrates excess moisture is removed by centrifugation or ultrafiltration to a protein concentration of 1.20-3.50 percent. By centrifugation at 3000 rpm receive a base with a concentration of 1.50%. Intensive centrifugation increases the concentration of the basics. The result is a gel-like collagen-based, consisting of thin fibrils without reactive groups, retaining its fluidity when radiation sterilization.

The formation of fibrils occurs with increasing pH of the solution to the value 4,30-5,30 adding alkali under vigorous stirring. To impart insolubility fibrils additional structure 0.1% solution of glutaraldehyde. Adding glutaraldehyde leads to the formation of intermolecular bridges that gives them resistance to the action of liquids and cells. According to the viscosity of denaturation structured fibrils occurs when 55oC, flat - 42oC. Washing with water to remove the excess aldehyde from suspension. However, for complete removal of reactive ALD of periodate sodium and other turns aldehyde groups to carboxyl with a small irritant. After processing, the excess moisture is removed by centrifugation, the basis Packed in plastic containers and sterilized by irradiation.

The principle of obtaining sterile bases from acidic solutions of collagen suitable for prolonged dosage forms of biologically active substances. Co-deposition of collagen, acidic glycosaminoglycan, glycoproteins, chitosan, etc. contributes to the immobilization of biologically active substances in the structure of collagen fibrils and subsequent cross-linking of slow bioresorption of collagen and release of biologically active substances, medicinal substances. In the interaction of the acidic solution of collagen, heparin, chondroitin sulfate, etc. in the range of pH 4,30-5,00 besieging fibrillar aggregates, which after treatment with 0.5% formaldehyde or 0.15% glutaraldehyde, there has been a gradual release of heparin within 30 days in the blood plasma.

In fibrillar basis optionally, you can include drugs with different pharmacological action and get soft dosage forms for external and internal use. Drugs should be ontouchevent; when added to a base of up to 50% powdered hydroxyappatite (hydroxypoly) get a plastic pasta stable after radiation sterilization. Pasta used to stimulate the processes of osteosynthesis.

In the basis of thin fibrillar collagen fibrils are in a swollen condition, have a high fluidity and easily pass through the needle of the syringe with a diameter of 0.5 to 2.0 mm. They are used in various fields of plastic surgery to fill cavities and defects of the connective tissue (tennova space, to fix the position of the vocal cords, wrinkles and so on ). Fibrillar framework can be used for the preparation of ointments, liniments, suspensions of various drugs. It can also be used for the manufacture of dry composite materials, sponges films after appropriate drying, etc.

Examples of specific implementation method.

Example 1. 1. To 1 l of a 0.6% solution of collagen in 0.2 M acetic acid /homogenizer RT 1/ added gradually with stirring, 1.5 g of hexamine dissolved in 100 ml of water. The mixture is homogenized for 1 h

2. After 1 h to separate the fibrils by centrifugation.

3. F. the fool was repeated 3 times to remove urotropine and products of its decomposition.

4. Washed fibrillar basis centrifuged at 6000 rpm for 30 min to obtain a protein concentration of 18-22 mg/ml, Packed in plastic tubes and sterilized by irradiation dose of 25 kGy.

Example 2. 1. To 1 l of acetic acid solution of collagen in 0.2 M acid in the mixer RT-1 add 2% solution of 2-substituted sodium phosphate mixture to pH 5.0 and after 10 min a solution of glutaraldehyde to its concentration in a mixture of 0.10%. After incubation over night at room temperature to separate fibrillar basis by centrifugation.

2. Rigorous 3-fold washing with 500 ml of water base to the absence in the lavage glutaraldehyde.

3. Processing of fibrillar 3% hydrogen peroxide solution for 2 h at room temperature.

4. 2-fold rinsing fibrillar basis to remove excess hydrogen peroxide.

5. Fibrillar basis subjected to centrifugation at 4000 rpm for 30 min to a concentration of 15 mg/ml

6. 300 g basis introduced gradually 1.5 g of hydroxyapatite powder and mix to obtain a homogeneous pasty mass white /pasta with hydroxyapatite/.

7. Packing pastes is in 0.3% acetic acid in the mixer RT-1 add 0.1 g of chondroitin sulfate in 50 ml of water and increase the pH of the mixture of 2-substituted phosphate sodium /5% solution to the magnitude of 4.4. After 10 min the mixture was added a solution of glutaraldehyde to 0.10% concentration and incubated at room temperature over night.

2. Carefully wash the mixture with water to remove excess moisture by centrifugation.

3. Further on PP 3-7 example 2.

Example 4. 1. Fibrillar basis obtained in examples 1, 2 and 3 is diluted with water to a concentration of 7-9 mg/ml and poured into a flat pan with a layer thickness of 3 or 6 mm, frozen and subjected to freeze-drying under vacuum.

2. Dry the plate material is cut into samples of respective sizes /for example 5x10 cm, Packed in plastic bags and sterilized by irradiation dose of 25 kGy.

Example 5. 1. To 1 l 0,40% solution of collagen in 0.2 acetic acid in the mixer RT 1 add 200 mg (5%) of chondroitin sulfate dissolved in 100 ml of water, and then 2% solution of 2-substituted sodium phosphate to pH 4.7 and after 10 min a solution of glutaraldehyde to a concentration of 0.10%.

After incubation for 8 h, as in PP. 2-5 example 2, separate the fibrillar mass, washed with water, treated with hydrogen peroxide, again washed with water and separated by centrifugation fibrillar mass.

2. To 300 g of fibrillar osnovnoi shell until a homogeneous paste-like mass of white.

3. Packing in plastic tubes and sterilized by gamma-irradiation dose of 25 kGy.

Example 6. 1. To 300 g of fibrillar substrate with a protein concentration of 2%, obtained in accordance with example 1, 2 or 3 with stirring, gradually add 0.3 g (5% by weight protein) methyluracil, resistant to irradiation, to obtain a homogeneous pasty mass.

2. Packing in plastic tubes and sterilize by irradiation dose of 25 kGy.

Literature

1. The ROM. patent N 88838, A 61 K 31/13, 31.03.86/25.11.83

2. Pat. USA, N 3949073, 424/177, 18.11.74/06.04.78.

3. Pat. USA, N 4582640, 260/123.7, 08.03.82/15.04.86.

1 1. A method of obtaining a collagen base from acidic solution of collagen, characterized in that use acetic acid solution deleteoperation collagen, which under intensive stirring, methenamine, or a solution of alkali metal salts mixture to pH 4,3 - 5,3, washed with water drawn fibrils, remove excess liquid phase to a protein concentration of 1.20 - 3.50 percent, filled pasty gel-like basis in polymer packaging is sterilized by gamma-irradiation dose of 25 kGy. 2 2. The method according to p. 1, characterized in that prior to the stage of washing water allocated fibrils process of 0.10% concrete excess liquid phase. 2 3. The method according to p. 1, characterized in that the selection of the collagen fibrils produced in the presence of up to 10% of the acidic collagen glycosaminoglycan is chondroitin sulfate, heparin, or chitosan. 2 4. The method according to p. 1 or 2, characterized in that the fibrillar basis enter up to 60% of a powder of hydroxyapatite or bone meal. 2 5. The method according to p. 1 or 2, characterized in that the fibrillar basics enter up to 20% of drugs that are resistant to sterilization by gamma-irradiation.

 

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