Viscoelastic gels as new fillers

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine. Described are biomaterials prepared by mixing an autocross-linked derivative of hyaluronic acid with hyaluronic acid derivative cross-linked with 1,4-butandiol diglycidyl ether (BDDE) in a weight ratio from 10:90 to 90:10, as new fillers.

EFFECT: biomaterials make is possible to promote the immediate regeneration/restoration of dermal/skin tissue, which has lost its initial tightness.

7 cl, 2 dwg, 16 ex

 

The technical field to which the invention relates.

Hyaluronic acid (HA) is heteropolysaccharide, consisting of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine.

HA is remotemachine polymer with a molecular weight in the range from 50,000 to 13×106Yes, depending on the source from which it is derived, and used methods to retrieve.

HA is present in nature in extracellular gels, the main substance of the connective tissue of vertebrates (one of the main components of which she is), in the vitreous body and the umbilical cord.

HA plays an important role in the biological organism as structural and mechanical support to tissues and as an active component in the cellular physiology of tissues, such as skin, tendons, muscles and cartilage.

It is one of the key molecules in the cartilage matrix, and is not the main protein constituent component of synovial fluid. Because it is a highly viscoelastic hydrophilic molecule, it gives synovial fluid lubricating properties; therefore HA was used for osteoarthritis for more than 30 years, mainly for the treatment associated with osteoarthritis pain.

HA also plays a key role in the process of reparation TC is either from a structural point of view (the organization of the extracellular matrix and the regulation of its hydration) and as an enabling regulatory substances of a wide range of physiological processes, where specified polysaccharide acts directly and/or indirectly (a blood clot, phagocytic activity, proliferation of fibroblasts, neovascularization, re-epithelialization, and so on) (P. Weigel et al., J Theoretical Biol, 1986:219-234; Abatangelo G. et al., J Surg Res, 1983, 35:410-416; Goa, K. et al., Drugs, 1994, 47:536-566). As these properties have been known for a long time, HA is also used for producing dressings to treat wounds, ulcers and skin lesions of different origin.

Hyaluronic acid is also used as filler for wrinkles, folds and small depressed areas on the skin and to increase the volume of lips and cheeks, because she is immunologically inert, non-toxic, biodegradable and biologically rassassyvesa.

Treatment means on the basis of hyaluronic acid is indicated for correction:

volume and contours of the lips,

skin folds (such as nasolabial folds),

correction of facial contours (e.g., cheeks and chin),

wrinkles (for example, folds nazirenes and oral commissures),

wrinkles around the eyes,

fibrotic acne scars,

fibrous posttraumatic scarring,

of soft tissue defects,

scars after the operation.

Hyaluronic acid is not a long-term filler. This means that after the injection of the product pic is epano metabolized and absorbed into the body for periods of time, changing in accordance with the processed region and used type of drug. The effect of filling and increased volume (or decrease wrinkles) manifests itself immediately and lasts only a few weeks. The main products present in the market can be classified into the following categories based on the different time periods of their resorption:

quickly rassasyvanie fillers (2-3 months),

fillers with mid-term resorption (5-6 months),

fillers slow resorption (1 year), such as Restylane Sub Q (QMed, EP0839159).

In the dermis HA performs hydrating functions due to its high ability to bind water and structural functions as a "frame", because by linking to other substances, it forms a macromolecular complexes, which give the skin density.

Therefore, the mechanism of action is immediate volumetric filling due to the viscoelastic properties of the product and the synthesis of new collagen due to stimulation of dermal fibroblasts.

However, HA is a natural polysaccharide which is quickly destroyed by the enzyme hyaluronidase present in connective tissue; in order to get the fillers, the effect of which lasts for a few months, HA therefore subjected to processes of cross-linkage,which improve its viscoelastic properties and increase the time it stays in the tissue. Educated so fillers are Poperechnaya, for example, by means of BDDE (simple diglycidylether ether of 1,4-butanediol, Restylane®, BELOTERO® and Regenyal Idea) or DVS (diphenylsulfone, Hylaform®), which create a bridge of communication between the molecules of the polymer. However, increasing the degree of cross-linkage causes progressive denaturation of HA to the extent deep modification of its chemical, physical and biological properties. Matrix of too poperechnogo HA are present in the form of solid substances in the form of particles that are no longer recognized by the cells (and, in particular, the immune system) as HA; therefore, the polysaccharide is perceived as a foreign body, which triggers an inflammatory reaction with the formation around him fibrational capsules. In addition, excessively Poperechnaya HA is not able to stimulate regeneration of the dermis/skin tissue, caused, as is known for a well-established scientific results, fragments of HA (particularly fragments with low molecular weight), which have the effect of stimulation of collagen synthesis in skin fibroblasts.

Fillers are also classified as rassasyvanie or long-term. Fillers rasskazyvaemoe type are the most biologically compatible; they consist of hyaluronic acid or collagen, or modified, or there is them in their natural form, and subsequently dissolve within most of the year. Long-term type consists of synthetic polymers such as polyacrylamides, special Poperechnaya molecules which form a stable gel when combined with water. Fillers long type always remain in situ and are very useful for filling the lips, but their use is not recommended, because their introduction into the skin more often causes an acute inflammatory reaction, leading to formation fibrocycstic capsules around filler, which are perceived as a foreign body and therefore are toxic.

The author has perfected a new type of biomaterial as a new filler and/or as a new product for profiling of the body, formed by mixing two Poperechnaya derivatives of HA in different but complementary ways to get Deputy leather/fabric, which provides immediate hydration (and therefore, immediate fill) processed leather/fabric, at the same time, contributing to very long periods of destructionin vivoto eliminate the need for repeated injections and, thus, reducing the side effects.

New biomaterials, belongs to the present invention, particular characteristics of the biological compatibility with identical characteristics who am hyaluronic acid as such, but their Biodegradability is different; with implantation ofin vivotheir presence in the body much longer than that of unmodified HA, thus enabling immediate recovery/restore dermal/skin tissue that has lost its original density.

Detailed description of the invention

The author has perfected a new type of biomaterial as a new filler and/or as a new product for profiling of the body based on the mixing of two derivatives of HA with different but complementary characteristics. To obtain a new product for injection in the treatment of skin defects in dermatology, cosmetic dermatology and/or aesthetic surgery, which causes:

1)immediate hydration of the dermis/skin,

2) the immediate filling of the treated tissue,

3) very long periods of destructionin vivo

4) reduced side effects.

New biomaterials consist of:

avtoperevozchiki hyaluronic acid (ACP) or hexadecylamine HA (HYADD) mixed with

hyaluronic acid, poperechnogo with BDDE (HBC).

ACP used in the present invention, obtained as described in EP 0341745, has a high degree of cross-linkage from 4 to 5% and preferably obtained using HA with an average molecular mass (MM) 200 kDa. When the Hydra is then it is present in the form of avtoperevozchikov gel without molecules, foreign to the native polysaccharide, because it arises from the ester bonds between the carboxyl and hydroxyl groups of the same polysaccharide chain and/or adjacent chains. It is therefore deprived of immunotoxicity, biologically compatible as native HA, has a high moisturizing ability and is easily destroyed by hyaluronidase, releasing a molecule with a low molecular weight, is capable of stimulating the synthesis of collagen, improve tone and elasticity of skin tissue.

Hexadecylamine HA (HYADD) receive, as described in EP 1095064 and EP 1853279, preferably using HA with a molecular mass (MM) 500-730 kDa, with an average final degree of amidation/substitution of 1 to 3 mole%.

ACP and HYADD are derived HA responsible for immediate hydration (leading to instantaneous filling of the dermis), called vnutriaortalina injection filler, relates to the present invention.

HA, Poperechnaya with BDDE (molecule containing epoxy group for the formation of ethers in primary hydroxyl HA), contains Poperechnaya molecule and therefore more resistant to enzymatic destruction, because it has essential links that stabilize the polysaccharide, providing the resulting product for a long time in the body.

Pach is the use of two types poperechnogo HA leads to the formation of a new biomaterial, which has the characteristics of biological compatibility, identical to the characteristics of native hyaluronic acid, but other bioraznolikost, so that after implantation ofin vivo, during his stay in the body much longer than the residence time of unmodified HA, thus providing the possibility of regeneration/repair of dermal tissue that has lost its original density. The author also demonstrated that their Union is unexpectedly leads to a much longer period of destructionin vivothan the time of the destruction of commercial control of fillers formed of the same type HA, poperechnogo with BDDE, with a subsequent increase residence time in the body. Finally, the author claims the priority of application of new biomaterials as fillers and/or as new products for profiling of the body in the treatment of skin defects in dermatology, cosmetic dermatology and/or aesthetic surgery.

Chemically heterogeneous nature of the new biomaterial provides the possibility of modulation of the properties of the final product by suitable variation of the ratios between the components. Two HA can be mixed in the ratio of ACP (or HYADD):HBC from 10:90 to 90:10; mass ratio will be selected based on the desired end elm the spine, which will depend on the treated area. If you have the processing stations requiring implantation of a large number of biomaterial, as in the case of filling Breasts, buttocks, cheeks or chin, or deep wrinkles, the biomaterial should preferably provide good density and, hence, viscosity, suitable to obtain a gel with excellent consistency and low speed bioraznolikost; in this case, a mixture of ACP (or HYADD):HBC will be from 10:90 to 50:50, and preferably 25:75, because the product is obtained by an increased mass fraction HBC, more appropriate to provide more long lasting increase the volume effect. However, if you're going to handle lip furrows or small forehead wrinkles, the ratio of the ACP (or HYADD):HBC preferably will be from 90:10 to 50:50, because a higher fraction of ACP in the filler ensures material more appropriate for biorevitalisation skin and correction of fine lines, wrinkles, small facial wrinkles, and the like. In addition, the needle should have a very high caliber; therefore, the gel should be easy squeezing and less viscous than the gel described above. The rheological properties of the product, therefore, can korrigirovanija based on the selected ratio ACP:HBC.

Its the tion of the biomaterial composed of a composition of equal quantities of ACP (or HYADD)/HBC may also appropriately be modulated by the target selection of the medium in which it is obtained: for example, a mixture mass ratio of ACP:HBC 50:50, dispersed in saline solution (0.9% NaCl), is more viscous than if she were dispersed in phosphate buffer at pH=6,95; therefore, for this particular mixture, the salt solution is more appropriate environment for making products with a limited speed dispersionin situ. Materials consisting of a predominant amount HBC, show the opposite profile. Consequently, the viscoelastic properties of the material affect product performance.

The present invention also relates to the two methods of obtaining the above biomaterials: methodAand the wayB.

New waysAandBdivided into two stages:

1) a method of deriving HBC and

2) the method of mixing it with the derived ACP or HYADD.

These two stages can lead to products with a very high degree of purity. When the methods commonly used to obtain HA, poperechnogo with BDDE, clean, do the washing mass of the obtained gel or dialysis. In both cases, the optimal cleaning efficiency may not be achieved due to the nature of m is tricy gel, which due to its tendency to swell, includes a large amount of solvent. These gels have low mobility and transport ability and tend to be precipitated in the form of gelatin resins. Thus obtained residue allocated in the form of a solid substance that has the properties of solubility and rheology during rehydration, in particular the ability to swell, elasticity and homogeneity (significant characteristics for filler) are different from the properties of the gel before cleaning.

However, in the method described below as method A, the product precipitates in the form of finely ground powder, which is then easily washed. In addition, careful selection of reaction conditions gives after separation by settling and washing the product with the ability recovery gel hydration and sterilization, providing a biomaterial having a reproducible, standardized enough characteristics of elasticity and uniformity.

WayBdoes not include the stage of precipitation of the product HBC in the form of a powder; cleaning and homogenization of the gel (obtained after mixing with HBC ACP or HYADD) perform on stage crushing, which includes passing it through a filter coefficient holding substances in the form of particles of from 25 to 150 μm. At this stage is the purification of the final gel that case is t his highly homogeneous.

HA used in the present invention for obtaining the above-described derivatives (HBC, ACP and HYADD), can be from any source, such as extraction from cocks ' combs or fermentation, and to have an average molecular weight of from 400 to 3×106Yes, preferably, from 1×105Yes to 1×106Yes, and more preferably from 200,000 to 1×106Yes.

A new way to getAincludes the following stages:

Synthesis poperechnogo HBC

1) Dissolving in an alkaline solution (preferably, 0.15 M and 0.35 M NaOH) diepoxides BDDE in the stoichiometric ratio of from 2.5 to 25 mole%, preferably from 5 to 15 mole% (depending on the intended use of the product; the higher the percentage of BDDE, the longer the residence time in the body) recurring units of hyaluronic acid with subsequent

2) the dispersion of HA in solution, described in the previous paragraph, at room temperature. The concentration of HA should be from 80 to 300 mg/ml, and time of homogenization from 30 to 300 minutes.

3) Start the reaction by thermal activation, and this solution is heated at a temperature of from 35 to 55°C for 2 to 36 hours.

4) Extrusion of the mass through a metal sieve to reduce the particles to a size of approximately 600 microns.

5) Hydration gel dilution with water by a factor of 3 to 25 during the time from 4 to hours at a temperature of from 4 to 24°C.

6) Bringing the pH to neutral values aqueous solution of HCl having a concentration of from 0.5 to 5 mol/l, preferably from 1 to 2 mol/L.

7) Add 2.5 volumes of water-soluble organic solvent, such as ethanol, methanol, isopropanol, n-propanol, dioxane, acetonitrile, acetone and/or mixtures thereof (preferably, ethanol and acetone), until, until there is obtained a product in the form of precipitated powder.

8) Washing with organic solvents, such as ethanol, methanol, isopropanol, n-propanol, dioxane, acetonitrile, acetone and/or mixtures thereof (preferably, ethanol and acetone) containing aqueous fraction below 35%.

9) drying in a vacuum at a temperature of from 30°to 45 ° C for 2 to 7 days, and in any case prior to the removal of residual solvents below the level of 400 hours/million of obtaining a white powder HBC.

Mixing ACP (or HYADD) with HBC

10) Mix the powder with HBC powder ACP (or HYADD) at a ratio of ACP:HBC from 10:90 to 90:10 (depending on the selected application, as described earlier).

11) Hydration with saline or phosphate buffer, preferably a saline solution (which may contain other excipients, such as lidocaine), leading to a total concentration of HA from 12 to 27 mg/ml, preferably, from 20 to 25 mg/ml, at a temperature of from 0 to 26°C.

12) Extrusion through a sieve with a mesh size of 50 to 500 μm, predpochtitelno, from 100 to 250 μm. The specified filtering performed at room temperature or at a temperature of from 25 to 65°C, preferably from 40 to 60°C.

13) Fill the syringe, preferably made of glass or polymer material obtained by the product.

14) Heat sterilization with saturated steam at a temperature of from 120 to 124°C (preferably, to 121.5±1°C) for at least 10 minutes

A new way to getBincludes the following stages:

Synthesis poperechnogo HBC

1) Dissolving in an alkaline solution (preferably, 0.15 M and 0.35 M NaOH) diepoxides BDDE in the stoichiometric ratio of from 2.5 to 25 mole%, preferably from 5 to 15 mole% (depending on the intended use of the product) of the repeating units of hyaluronic acid, with subsequent

2) the dispersion of HA in solution, described in the previous paragraph, at room temperature. The concentration of HA should be from 80 to 300 mg/ml, and time of homogenization from 30 to 300 minutes.

3) Start the reaction by thermal activation, and this solution is heated at a temperature of from 35 to 55°C for 2 to 36 hours.

4) Bringing the pH to a neutral aqueous solution of HCl having a concentration of from 0.05 to 1 mol/l, preferably 0.1 mol/L.

5) Hydration gel dilution with water by a factor of 3 to 20 over a period of time from 4 to 48 hours at a temperature of from 4 d is 24°C. This solution may contain other excipients, such as NaCl, sodium and potassium salt of phosphoric acid and lidocaine, preferably in the form of hydrochloride. Sodium salts (chloride and phosphate) keep appropriate osmolarity of the product and maintain the pH at values comparable to the pH of the tissues. In a preferred embodiment of the invention NaCl is added in an amount such that the final solution contained him in a concentration of from 0.8 to 1.0%, preferably, 0.9 per cent; hydrochloride lidocaine, in his presence, is added in an amount such that the final composition contains it in an amount of from 2.2 to 3.2 mg/ml, preferably, 2.7 mg/ml

Mixing ACP (or HYADD) with HBC

6) Mixing the gel with HBC powder ACP (or HYADD) at a ratio of ACP:HBC from 10:90 to 90:10 (by weight of active ingredient) depending on the application, the new filler, as described above. Alternatively, ACP or HYADD can be mixed with HBC, since both components in the form of a gel, using a suitable mixing device (preferably, orbital blade stirrer) during the period of time from 30 minutes to 24 hours at a temperature of from 0 to 26°C.

7) Crushing and homogenization passing through the filter coefficient holding substances in the form of particles ranging from 25 to 150 μm, preferably from 40 to 110 MK is. If the viscosity is excessive, the operation can be performed at a high temperature from 25 to 65°C.

8) Filling syringes manufactured for glass or polymer material obtained by the product.

9) Sterilization is heated by saturated steam at a temperature of from 120 to 124°C (preferably, to 121.5±1°C) for at least 10 minutes

Some examples of obtaining a new filler in accordance with the invention are described below as examples, but not limitations.

Example 1: Synthesis of HBC 500 (HA 500-730 kDa)

The way A

0,075 mol HA with a molecular mass of 500-730 kDa, obtained by fermentation is dispersed in 215 ml of 0.25 M NaOH containing 1,41 ml BDDE. The mixture is then heated to 42°C and subjected to interaction within 3 hours. The mixture is then hydratious for 24 h with 300 ml of solution containing stekhiometricheskie amount of HCl to bring the pH to neutral. The total volume was adjusted to 750 ml and precipitated with 2.5 volumes of ethanol to obtain a filtered remove sediment. The mixture was washed with 75% ethanol until complete precipitation, verified by measuring the conductivity of the washing solvent, which must be less than 30 µs/cm, and dried in vacuum at 40°C for 5 days. Product HBC get with the mass release of 87%.

Example 2: Synthesis of HBC 1000 (HA 1 MDA)

The way A

1,60 g HA with an average molecular what assay 1 MDA, obtained by fermentation, was dispersed in 20 ml of 0.25 M NaOH solution containing 75 μl of BDDE. The mixture is then heated to 42°C and subjected to interaction within 2 hours. The mixture is then hydralicious within 24 h in 20 ml of a solution containing stekhiometricheskie amount of HCl to bring the pH to neutral. The total volume was adjusted to 75 ml and HBC precipitated with 2.5 volumes of ethanol to obtain a filtered remove sediment. The mixture was washed with 75% ethanol until complete cleaning, verified by measuring the conductivity of the washing solvent, which must be less than 30 µs/cm, and dried in vacuum at 40°C for 5 days. Product HBC 1000 get with the mass release of 90%.

Example 3: Synthesis of HBC 200 HA 200 kDa)

The way A

2,55 g HA with an average molecular weight of 200 kDa, obtained by fermentation, was dispersed in 20 ml of 0.25 M NaOH solution containing 63 μl of BDDE. The mixture is then heated to 42°C and subjected to interaction within 150 minutes. The mixture is then hydratious within 24 h in 20 ml of a solution containing the stoichiometric amount of HCl. The total volume was adjusted to 75 ml and precipitated with 2.5 volumes of ethanol to obtain a filtered remove sediment. The mixture was washed with 75% ethanol to achieve a thorough cleansing, verified by measuring the conductivity of the washing solvent, which must be less than 30 µs/cm, and dried in vacuum at 4°C for 5 days. Product HBC 200 get with the mass release of 85%.

Example 4: preparation of a gel ACP:HBC 500 in the ratio of 50:50

The way A

1,00 g HBC 500, obtained as described in example 1, is mixed with a 1.00 g internal of ester HA ACP. Powder hydratious 100 ml of 0.9% wt./about. sterile saline solution at a temperature of 8°C for 16 hours. The obtained gel is heated to 48°C, filtered through a metal sieve with a cell size of 0.17 mm and then distribute between glass syringes 1 ml, which are then subjected to sterilization with saturated steam at 121°C for 10 minutes. Get homogeneous sterile gel, suitable for local administration.

Example 5: preparation of a gel ACP:HBC 1000 in the ratio 30:70

The way A

1.40 g HBC 1000, obtained as described in example 2, is mixed with 0,60 g internal of ester HA ACP. Powder hydratious 100 ml of 0.9% wt./about. sterile saline solution at a temperature of 8°C for 16 hours. The obtained gel is heated to 48°C, filtered through a metal sieve with a cell size of 0.17 mm and then spread on glass syringes with a capacity of 1 ml, which are then subjected to sterilization with saturated steam at 121°C for 10 minutes. Get homogeneous sterile gel, suitable for local administration.

Example 6: preparation of a gel ACP:HBC 500 in soothes the NII 25:75

The way A

1,875 g HBC 500, obtained as described in example 1, is mixed with 0.625 g of internal complexity ether HA ACP. Powder hydratious 100 ml of 0.9% wt./about. sterile saline solution at a temperature of 8°C for 16 hours. The obtained gel is heated to 48°C, filtered through a metal sieve with a cell size of 0.19 mm and then poured into glass syringes with a capacity of 1 ml, which are then subjected to sterilization with saturated steam at 121°C for 12 minutes. Get homogeneous sterile gel, suitable for local administration.

Example 7: preparation of a gel ACP:HBC 1000 in the ratio of 75:25

The way A

0.50 g HBC 1000, obtained as described in example 2, is mixed with 1.50 g of internal complexity ether HA ACP. Powder hydratious 100 ml of 0.9% wt./about. sterile saline solution at a temperature of 8°C within 24 hours. The obtained gel is heated to 42°C, filtered through a metal sieve with a cell size of 0.17 mm and then poured into glass syringes 2 ml, which are then subjected to sterilization with saturated steam at 121°C for 12 minutes. Get homogeneous sterile gel, suitable for local administration.

Example 8: preparation of a gel HYADD:HBC, in the ratio of 60:40

The way A

1.20 g HBC 500, obtained as described in example 1, is mixed with 0,80 g hexadecylamine HA (HYADD).Powder hydratious 100 ml of 0.9% wt./about. sterile saline solution at a temperature of 8°C within 24 hours. The obtained gel is heated to 52°C, filtered through a metal sieve with a cell size of 0.17 mm and then poured into glass syringes with a capacity of 1 ml, which are then subjected to sterilization with saturated steam at 121°C for 11 minutes. Get homogeneous sterile gel, suitable for local administration.

Example 9: preparation of a gel HYADD:HBC 500 in the ratio of 40:60

The way A

8.0 g of sodium salt of HA with an average molecular weight of 500-730 kDa, obtained by fermentation, was dispersed in 40 ml of 0.25 M NaOH solution containing of 0.44 ml of BDDE. The mixture is heated at 41.5°C for 2 hours and 40 minutes. Then it hydratious during the night 100 ml of 0.1 M HCl solution and 200 ml of water. Add 50 ml of saturated NaCl solution, and the mixture is left to swell overnight. The next day, add 170 ml of acetone and 30 ml of saturated NaCl solution, and the mixture is precipitated by slow addition of one liter of ethanol. The precipitate was washed with the same solvent to remove residual NaCl, then dried in an oven at 35°C under vacuum until then, until the residual solvents are removed. Thus obtained powder HBC mixed in the ratio 5:3 with HYADD, obtained as described in patent EP 1853279. Mixed powders hydratious salt solution, leading to a total concentration of 20 mg/ml (with the relevant 12.5 mg/ml HBC and 7.5 mg/ml HYADD4). The product is left to swell at 5°C and the next day filtered through a flat membrane with a nominal degree of retention of 100 microns. Glass syringes 1 ml fill the thus obtained product and sterilized in a loop with F0=13 when to 121.5°C.

Example 10: the Filling of the skin and tolerability gel HYADD:HBC model vnutricerepnogo the introduction of rabbits

The aim of the experiment was to evaluate the filling of the skin, revealing the beginning of any macroscopic side effects and tissue reaction caused by the gel HYADD:HBC (obtained as described in example 9), injected in vnutriaortalina fabric rabbit, by comparison with a commercial filler BELOTERO®.

For the evaluation, test gels were injected vnutriaortalina male NZW rabbits-KBL weighing from 1.8 to 2.3 kg

The structure of the experiment:

Animals were anestesiologi intravenous ketamine and xylazine. For each test filler used 3 animals.

Day 0:T0

injection of samples (1 ml of hydrogel on the sample) after shaving the backs of rabbits;

measurement of swelling in rabbits and macroscopic observation to identify side effects.

Day 7:T7

Measurement of the volume of swelling and macroscopic observation to identify side effects.

The amount of swelling was calculated by the formula:

(2/3×π)×(r1)×(r2)×(r3

where: (r1), (r2) and (r3) are respectively the width, length and height of the swelling measured by the compass.

Results:

New filler did not cause any inflammation in the treated dermis.

The results obtained for the residence time in the tissue, as shown in Fig.1: quantification of swelling, estimated in the first week of treatment (expressed as mm3) demonstrated that the gel according to the invention is able to cause greater than in the control, the amount of swelling of the skin, which remained high even after 7 days, also to a much greater extent than commercial filler, used as a comparison. These data show clearly that the new fillers immediately cause significant hydration of the dermis, and this effect can be attributed to the presence of derived HYADD, which due to its chemical/rheological characteristics were essential to facilitate the immediate filling of the skin, which remains stable over time.

Example 11: Synthesis of HBC 500 (HA 500-730 kDa)

Method B

18.75 g of sodium salt of HA with a molecular mass of 500-730 kDa, obtained by fermentation, is dispersed in 133 ml of 0.25 M NaOH solution containing 885 μl of BDDE. The mixture is then heated at 45°C for 2.5 hours. The mixture hydratious during the night of 0.62 l of a solution containing CTE is americasee amount of HCl, 2.65 g of NaCl and 2.7 g of the hydrochloride lidocaine, with slow stirring.

Example 12: preparation of a gel ACP:HBC 500 in the ratio 25:75

Method B

6.25 g of internal ester of hyaluronic acid ACP 200 solubilizer in 250 ml of a solution containing 4.4 g of NaCl, with slow stirring. When hydration is complete, the gel is combined with the gel obtained in accordance with example 11, in the mixer, equipped with a device for mixing the semi-solid substances to homogeneity. The obtained gel is extruded through a flat membrane filter with a nominal degree of retention of the substance in the form of particles 70 μm. Thus obtained product is introduced into glass syringes and sterilized in a loop with F0=13 when to 121.5°C.

Example 13: preparation of a gel HYADD:HBC 500 in the ratio 25:75

Method B

6.25 g of hexadecylamine HYADD solubilizer in 250 ml of a solution containing 4.4 g of NaCl, with slow stirring. When hydration is complete, the gel is combined with the gel obtained in accordance with example 11, in a mixer equipped with orbital mixing device until smooth. The obtained gel is extruded through a flat membrane filter with a nominal degree of retention of material in the form of particles 70 μm. Thus obtained product is introduced into glass syringes and sterilized in a loop with FO=13 when to 121.5°C.

Example 14: Synthesis 500 BC (HA 500-730 kDa)

Method B

125 g of sodium salt of HA with a molecular mass of 500-730 kDa, obtained by fermentation, is dispersed 1.33 l of 0.25 M NaOH solution containing 9.4 ml BDDE. The mixture is heated at 45°C for 2.5 hours. The mixture hydratious during the night 6,2 l of a solution containing the stoichiometric amount of HCl, 26,5 g NaCl and 27 g of the hydrochloride lidocaine, with slow stirring.

Example 15: the precipitation of the gel ACP:HBC 500 in the ratio of 50:50

Method B

125 g of internal ester of hyaluronic acid ACP200 solubilizing 2.5 l of a solution containing 44 g of NaCl, with slow stirring. When hydration is complete, the gel is combined with the gel obtained in accordance with example 14, in a mixer equipped with orbital mixing device with a reflective baffle and scraper. The obtained gel is extruded through a flat membrane filter with a nominal degree of retention of material in the form of particles of 45 μm. Thus obtained product is introduced into glass syringes and sterilized in a loop with FO=13 when to 121.5°C.

Example 16: the Filling of the skin and tolerability of ACP gel:HBC model vnutricerepnogo the introduction of rabbits

The experiment was performed as described in example 10, using the gel obtained as described in examples 11-12, and by comparing it with the control Belotero® and with the second commercial filler, Regenyal Idea.

For the frame of the experiment, the author is not only determined the amount of swelling of the skin caused by processing, but also estimated the total residence time in the tissue gel/filler according to the invention by comparison with two well-known commercial fillers, which are the final comparative sample, because they both consist of HA, poperechnogo with BDDE.

Swelling of the skin in rabbits after treatment was measured every two weeks (with macroscopic observation to identify side effects) for up to 96 days.

Results:

In Fig.2 shows the results obtained were confirmed by the above data, namely immediate hydration processed dermis (mainly during the first 7 days) in a surprisingly greater extent than in controls; in addition, the amount of swelling of the skin was clearer, and the residence time in the tissue longer than the figures from the two commercial samples of comparison. At the end of the experiment a new filler according to the invention were still present, while both the control sample had almost disappeared.

It should be understood that the methods described herein, can be modified in various ways. Such modifications should not be construed as departing from the nature and prospects of the invention, and all modifications that might seem obvious is innymi for professionals in this field, included in the scope of the following claims.

1. Biomaterials obtained by mixing
avtoperevozchikov derived hyaluronic acid (ACP) with
derived (HBC) hyaluronic acid, Poperechnaya with simple diglycidyl ether of 1,4-butanediol (BDDE),
mass ratio of from 10:90 to 90:10, as new fillers and/or quality of products for the profiling of the body.

2. Biomaterials under item 1, preferably in a weight ratio of from 90:10 to 50:50, as biorevitalisation fillers.

3. Biomaterials under item 1 in weight ratio of from 10:90 to 50:50 with the effect of increasing the volume.

4. Biomaterials under item 3, preferably in a mass ratio of 25:75.

5. Biomaterials under item 1, additionally containing a carrier, where the carrier is a saline solution.

6. Biomaterials under item 1, additionally containing lidocaine.

7. Biomaterials under item 5, additionally containing lidocaine, where the carrier comprises saline solution.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: method of production of the water-soluble bioactive nanocomposite comprising salt of hyaluronic acid modified by citric acid or salt of citric acid, as a matrix, and gold nanoparticles as the filler is carried out by chemical interaction of solid-phase powders of salt of hyaluronic acid, citric acid or salt of citric acid and aurichlorohydric acid or salt of gold under the temperature from -18° to 125°C, under conditions of simultaneous action of pressure from 50 to 1000 MPa and the shear deformation in a mechanochemical reactor.

EFFECT: invention enables to obtain water-soluble bioactive nanocomposite with reliably predictable characteristics, namely the high yield of the target product, high gold content, controlled size of gold nanoparticles, narrow distribution by size of nanoparticles of gold, significant increase in resistance of the composite during long storage the above parameters of the composite are maintained for at least one year, the method is carried out in the absence of the liquid medium and does not require the stage of purification and concentration.

20 cl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chitosan carboxyalkylamide hydrogel and may be used for cosmetic and dermatological treatment of skin burns. Chitosan carboxyalkylamide hydrogel of pH close to that of skin and making 6.5 to 7.2 contains 40 to 90 mole % of the groups of N-carboxyalkylaminde D-glucosamine of formula (I) wherein n represents an integer 1 to 8, 60 to 10 mole % of the protic groups of D-glucosamine, and 5 to 15 mole % of the groups of N-acetyl-D-glucosamine. A method for preparing said hydrogel involves preparing an acid solution of chitosan of a degree of acetylation of 85 to 95%, providing a reaction of produced additive chitosan salt in an aqueous solution of diorganic acid and correcting pH of the prepared solution.

EFFECT: preparing the anhydrous product of chitosan carboxyalkylamide prepared by hydrogel dehydration.

12 cl, 1 dwg

FIELD: chemistry.

SUBSTANCE: biocompatible, biodegradable porous composite material contains chitosan and hydrosilicate filler in amount of 0.05-10% of the weight of chitosan and has a system of through pores with size of 5-1000 mcm. The method of producing the material involves mixing hydrosilicate filler, which is pre-dispersed in an aqueous medium with pH=5-7 in an ultrasonic field with frequency v=20-100 kHz for 5-60 minutes, with chitosan in an amount which corresponds to its concentration in the solution of 1-4 wt %, the amount of the filler being equal to 0.05-10% of the weight of chitosan; the obtained mixture is then intensely mixed at temperature of 20-50°C for 20-60 minutes; concentrated acetic acid is added in an amount which enables to obtain, in the mixture of the aqueous solution, acetic acid with concentration of 1-3%; the mixture is intensely mixed at temperature of 20-50°C for 20-250 minutes and then cooled to temperature of -5 to -196°C; the solvent is removed in a vacuum; the obtained end material is treated with a neutralising agent, washed with water to pH=5-7 and then dried.

EFFECT: presence of a system of through pores and providing a stable porous structure of the material in aqueous medium, eliminating cytotoxicity.

6 cl, 9 ex, 6 dwg

FIELD: chemistry.

SUBSTANCE: biodegradable film contains pectin, chitosan, water, 1N hydrochloric acid, a plasticiser - glycerine and a structure-forming agent - 3% methyl cellulose solution.

EFFECT: obtaining a uniform biodegradable film without structural defects, which similar to household packaging polyethylene films on homogeneity, plasticity and strength.

2 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to synthetic polymer chemistry. The nanocomposite contains a matrix in form of a cross-linked salt of hyaluronic acid which is modified with sulphur-containing compounds and nanoparticles of a noble metal as filler. A film of the cross-linked salt of hyaluronic acid which is modified with sulphur-containing compounds is obtained through chemical reaction of the salt of hyaluronic acid with a mixture of two sulphur-containing compounds and with a cross-linking agent, under conditions with pressure between 50 and 300 MPa and shear deformation in a mechanical reactor at temperature between 20 and 30°C. The reactor used to obtain the film is a Bridgman anvil.

EFFECT: invention enables to obtain a range of new bioactive nanocomposites with quantitative output and in the absence of a liquid medium, where the method does not require high energy, labour and water consumption and significantly increases efficiency of the composite; in particular, resistance to decomposition in the presence of hydroxyl radicals is 2-3 times higher compared to the control result.

16 cl, 7 ex

FIELD: medicine.

SUBSTANCE: solution of viable breast cells after enzymatic degradation in a collagenase solution of analysed tissue at temperature 37°C for 30-35 minutes, are applied on a biomaterial of a native form of hyaluronic acid, stored at room temperature and constant humidity 30-50 %, and cell viability is controlled by discoloration of cell monolayer.

EFFECT: invention allows producing a viable cell monolayer and providing their vital activity for 2-3 hours.

1 tbl, 2 dwg

FIELD: chemistry.

SUBSTANCE: articles based on biologically active polymer materials can be used in surgery when treating wounds and as materials for temporary replacement of body tissue, in biotechnology for obtaining matrices for growing cell cultures, in pharmaceuticals as carriers of enzymes and other biologically active compounds. The cross-linking reagents are 2,4-derivatives of 3-oxa glutaric dialdehyde (2,2'-oxydiacetaldehyde). These compounds can be obtained via periodate oxidation of monosaccharides, nucleosides and nucleotides.

EFFECT: low toxicity and biocompatibility of chitosan-based materials is achieved by using cross-linking reagents.

2 dwg, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of obtaining polysaccharide fibre for making materials, specifically for making surgical suture materials absorbable in a human and a mammal body, absorbable and non-absorbable dressing material and absorbable textile matrix materials. The method is characterised by that 2.4-4.0 wt % solution of polysaccharide in dimethylacetamide which contains 4.56-10.00 wt % lithium chloride is mixed with 1.0-5.0 wt % poly-N-vinylpyrrolidone with molecular weight of 8-35 kDa or a metallopolymer complex - fine-grained silver stabilised by poly-N-vinylpyrrolidone in such an amount that, content of fine-grained silver relative polysaccharide dissolved in spinning solution ranges from 0.07 to 0.87 wt %. Mass ratio of polysaccharide: metallopolymer complex equals 88.0-99.0:1.0-12 wt %. The mixture is intensely stirred, held, filtered, degassed and the obtained spinning solution is extruded at room temperature into an alcohol deposition tank in form of water-soluble aliphatic C2 and C3 alcohols. The fibre is then processed in plastification and washing tanks and dried.

EFFECT: obtaining fibre with good deformation and strength properties.

1 dwg, 4 tbl, 29 ex

FIELD: chemistry.

SUBSTANCE: invention relates to taxane, especially to paclitaxel and docetaxel covalently bonded with hyaluronic acid or a hyaluronic acid derivative, used as active substance in pharmaceutical compositions which are used in oncology, for treating tumours, autoimmune diseases and restenosis, as well as a coating for stents and medical devices. The covalent bond is formed between hydroxyl groups of taxane and carboxyl groups or hydroxyl groups of hyaluronic acid or hyaluronic acid derivatives, or amino groups of deacetylated hyaluronic acid. Bonding can take place using a linking compound (spacer compound) which bonds taxane with hyaluronic acid or hyaluronic acid derivative under the condition that, the linking compound is different from hydrazide.

EFFECT: proposed taxane has high therapeutic effectiveness when treating oncological diseases, autoimmune diseases and restenosis, dissolves in water without reduction of its pharmacological activity and is not toxic, which leads to overcoming hypersensitivity and anaphylaxis.

46 cl, 20 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: composition includes chitosan gel, which has bactericidal properties, representing matrix for including in it of water solution for "Adgelon" injection, which contains serum glycoprotein from blood of livestock, possessing biological activity in supersmall doses 10-9-10-15 mg/ml, and calcium salt - preparation "Adgelon". In obtaining composition medicine "Adgelon" is immobilised on chitosan gel.

EFFECT: composition provides increased ability to stimulate proliferation of osteoblasts, reparative processes of osteoformation with recovery by means of morphologically normal bone matrix.

2 cl, 3 ex

FIELD: medicine.

SUBSTANCE: there are described new reinforced biodegradable frames for soft tissue regeneration; there are also described methods for living tissue support, extension and regeneration, wherein the reinforced biodegradable frame is applied for relieving the symptoms requiring high durability and stability apart from patient's soft tissue regeneration. What is described is using the frames together with cells or tissue explants for soft tissue regeneration in treating medical prolapsed, e.g. rectal or pelvic prolapse, or hernia.

EFFECT: frames are adequately durable to be applicable for implantation accompanying the medical conditions requiring the structural support of the injured tissues.

14 cl, 19 dwg, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and tissue engineering, and may be used in cardiovascular surgery for small-vessel bypasses. A vascular graft is made by two-phase electrospinning with the staged introduction of the ingredients into the polymer composition.

EFFECT: making the bioresorbed small-diameter vascular graft possessing the improved biocompatibility ensured by using the polymer composition of polyhydroxybutyrate (PHBV) with oxyvalerate, and epsilon-polycaprolactone with type IV collagen, human fibronectin and human fibroblast growth factor (hFGF) additionally introduced into the composition.

2 cl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to porous microsphere granules with the adjusted particle size for bone tissue regeneration. The above microspheres have a size within the range of 1-1000 mcm, have through pores of the size of 1-100 mcm and total porosity 40-75%. The declared microsphere granules are prepared by granulation by electrospinning, and heat-treated. A mixture used to form the granules by electrospinning contains a mixture of magnesium orthophosphate and biological hydroxyapatite of bovine demineralised bones in ratio 0.5:1.0, as well as 1-3% sodium alginate in distilled water and a hardener representing saturated calcium chloride.

EFFECT: invention provides preparing the microsphere granules possessing biocompatibility, biodegradation, osteoinduction and osteoconduction properties and able to be substituted by the bone tissue.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and tissue engineering, namely to cardiovascular surgery and may be used in coronary artery bypass surgery, as well as in surgical reconstruction of peripheral vessels. What is described is a method for making a porous tubular matrix of a vascular graft of a biodegradable polymer by two-phase electric spinning, with biologically active molecules stimulating the vascular regeneration being incorporated into a matrix wall matrix incorporated biologically active molecules.

EFFECT: creating the tissue-engineered high-patency and durability small-diameter vascular graft for biological re-modelling of the damaged vessels in vivo.

2 cl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine. What is described is an implanted multilayer chondral reparation flap showing biological compatibility and physiological resorption, and what is also described is a method providing surgical management in situ for intra-articular regeneration of cartilaginous tissue in joint damages and/or defects. The chondral reparation flap comprises a first external cell-impermeable layer and a second external cell-permeable layer adapted for placement in an immediate proximity from a subchondral bone on a wound portion, and also a cartilage-forming matrix located between the first and second layers. The cartilage-forming matrix represents an accepting medium for diffusion of autologous stem cells and contains chemical components promoting formation of a hyaline-like cartilage in the presence of said autologous stem cells. The method prevents a fibrous cartilaginous replacement tissue from forming within the injury region.

EFFECT: method provides autologous compositions which when used in a combination with the reparation flap form the medical system for formation of the replacement hyaline-like intra-articular cartilage.

17 cl, 7 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: porous matrix based on biocompatible polymer or polymer mix for tissue engineering is obtained by compression of polymer and sodium chloride particle mix with defined particle size, and further removal of sodium chloride by dissolution. Porosity grade of matrix lies within 93 to 98%, its pores fall into different sizes, with definite pore distribution by size within certain limits.

EFFECT: obtained matrices are free-shaped yet pertain stability and hardness characteristics required to withstand surgical implantation methods and counteract mechanical forces applied at the implantation point.

40 cl, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: present group of inventions concerns medicine, more specifically coated implants and devices. There is offered ceramic composition-precursor for making high-strength bio-elements used as an absorbable or partially absorbable biomaterial where the composition contains at least one silicate with Ca as a base cation with the absorption rate less or equal to the bone growth rate, and this at least one silicate acts as a base binding phase in a biomaterial, and this at least one silicate Ca is present in amount 50 wt % or more, and all other components if any are presented by additives, such as an inert phase, and/or additives which make a biomaterial to be radiopaque. There is offered hardened ceramic material which is based on the ceramic composition-precursor and is in the hydrated form. There is offered a medical implant, application of the medical implant, and also a device or a substrate coated with the uncured ceramic composition-precursor and/or hardened ceramic material.

EFFECT: invention provides a biomaterial having initial and constant durability which is dissolved in due time and reacts with an organism to generate a new tissue.

29 cl, 1 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine. Claimed is composition with hyaluronic acid (HA), which includes gel particles of bound water-insoluble hydrated HA. HA includes bindings, represented with the following structural formula: HK'-U-R2-U-TK'. Where each group HA' represents the same or other molecule of bound HA'; each U independently represents optionally substituted 0-acylisourea or N-acylurea; and R2 represents optionally substituted alkyl, alkenyl, alkinyl, alkoxy, cycloalkyl, cycloalkenyl, cycloalkinyl, aryl, heteroaryl, heterocyclic radical, cycloaliphatic alkyl, aralkyl, heteroaralkyl or heterocyclolalkyl. Also claimed is method of developing tissues in individual, including introduction of needle into individual in place where development of tissues is necessary, needle is connected to syringe filled with composition with HA, and applying force to syringe in order to supply composition with HA to individual. Method of obtaining composition with HA includes formation of water-insoluble dehydrated particles of bound HA, separating insoluble in water particles by their average diameter, selection of subset of particles by average diameter and hydration of subset of dehydrated particles by means of physiologically compatible water solution. Other method of obtaining composition with bound HA includes binding precursor of bound HA by means of bis-carbodiimide in presence of pH buffer and dehydration of bound HA. Also included is method of developing tissues in individual that needs tissue development. Method of stabilisation of bound HA includes hydration of water-insoluble dehydrated bound HA by means of physiologically compatible water solution which includes local anesthetic, so that value of elasticity module G' for stabilised composition constitutes not less than approximately 110% from value G' for non-stabilised composition.

EFFECT: claimed composition of hyaluronic acid and method of preparation and application of HA composition are efficient for development of tissue and/or drug delivery.

27 cl, 22 ex, 2 tbl, 7 dwg

FIELD: chemistry.

SUBSTANCE: effect is achieved by using compositions based on different stereoregular amorphous biodegradable polymers - polylactides and copolymers of lactides with glycolides (18-72 mass ratio) as the second component of biocompatible mineral filler - hydroxyapatite with particle size of the main fraction of 1-12 mcm (8-41 mass ratio), as well as an organic solvent with boiling temperature equal to or higher than softening temperature by 3-20°C (20-41 mass ratio). After preparation of a homogenous mixture, the composition is undergoes thermal treatment at 80-130°C in a vacuum in a shaping vessel with the required shape. A porous product is obtained due to removal of solvent. Density of the obtained porous product is about 0.4-0.8 g/cm3.

EFFECT: design of a method of obtaining porous biodegradable composite polymer products based on polylactides or copolymers of lactides and gylcolides.

3 cl, 3 ex

FIELD: medicine.

SUBSTANCE: described are implants based on biodegradable thixotropic compound with pseudo-plastic properties and implant injected under skin or into skin in fibrous tissue. Containing microparticles of at least one biocompatible ceramic compound in suspension, in at least one liquid carrier containing at least one compound based hyaluronic acid and at least one biodegradable thixotropic compound with pseudo-plastic properties. Also disclosed is kit for preparation such implants directly before application, as well as implant production and using for filling of crinkles, and/or skin cavity, and/or cicatrices.

EFFECT: implants of simplified injection.

14 cl, 4 ex

FIELD: medicine.

SUBSTANCE: there are described compositions containing hyaluronic acid with a low degree of modification of functional groups, and mixtures prepared by a controlled reaction of this slightly modified hyaluronic acid with applicable difunctional or polyfunctional cross-linking agents. The compositions possess the low anti-inflammatory properties in injection in vivo and can be used as medical devices, biomedical adhesives and sealing matters.

EFFECT: targeted delivery of the bioactive substances.

49 cl, 14 dwg, 24 tbl, 45 ex

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