Cross-linked hyaluronic acid and production method thereof
SUBSTANCE: method involves activation of hyaluronic acid using a cross-linking agent and an auxiliary cross-linking agent. The activated hyaluronic acid then reacts with a nucleophilic cross-linking agent. The pH of the reaction medium ranges from 8 to 12. The nucleophilic cross-linking agent contains at least 50 wt % oligopeptide or polypeptide. Further, pH of the reaction medium is regulated to 5-7 and cross-linked hyaluronic acid is precipitated in the organic solvent. The invention also relates to use of the cross-linked hyaluronic acid obtained using this method in plastic surgery to make implants and to a hedrogel containing said cross-linked hyaluronic acid in a buffer aqueous solvent.
EFFECT: invention enables to obtain cross-linked hyaluronic acid in dry form, having high resistance to decomposition factors such as temperature, free radicals and enzymes.
18 cl, 3 tbl, 3 ex
The present invention relates to a new poperechnogo hyaluronic acid, and method of its manufacture and its use, in particular for cosmetic purposes.
Hyaluronic acid is a polysaccharide composed of chains of D-glucuronic acid and parts of N-acetyl-D-glucosamine, which is known to be used in plastic surgery and eye surgery, as well as in cosmetology as a product to fill wrinkles. In the latter application, in particular, hyaluronic acid is preferred over other fillers because of its biocompatibility and physico-chemical properties. However, it has the disadvantage that it decomposes quickly, which requires repeated injections. To address this shortcoming suggested various ways cross-linkage of hyaluronic acid, designed to make it less sensitive to various factors of decomposition, such as enzymatic and/or bacterial exposure, temperature and free radicals, and thus, to improve its resistance to degradation in vivo and, therefore, increase the duration of its action. These methods include, in particular, esterification or amidation of hydroxyl and/or acid functions of natural hyaluronic acid.
Known methods of cross stitching hyaluronic is islote, in particular, amidation, however, have the disadvantage that arise derivatives of hyaluronic acid, which is difficult to enter into the aquatic environment and/or are not sufficiently resistant to the factors of the decomposition, in particular after sterilization of the product.
This applies to water-insoluble hyaluronic acid, produced according to the request of the U.S. 2001/0393369 by the reaction in the acidic environment of hyaluronic acid with an activator such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and the nucleophile, which can be polylysin.
In fact, it is believed that at pH below or equal to 7 expected amidation reaction is completed the reaction is intramolecular esterification, which leads to self-cross-linking of the primary alcohol of hyaluronic acid on the activated complex ester of hyaluronic acid. This parasitic reaction, in particular, is reflected in a significant increase in viscosity (zagustevanii) and the turbidity of the reaction mixture, which thus is in the form of a heterogeneous mixture of water and insoluble polymer. Then it becomes impossible to distinguish derived hyaluronic acid.
In addition, in the application EP-1535952 disclosed coating consisting of poperechnogo hyaluronic acid formed in situ by reaction of polylysine with hyaluronic acid in the presence of EDC and NHS at a pH of from 2 d is 9, preferably from 4 to 7.5. The product is equipped with such a coating may, in particular, to serve as a prosthesis in aesthetic surgery. This document does not disclose Poperechnaya hyaluronic acid, precipitated in an organic solvent, in order to get her dry and immediately be manufactured in the form of a hydrogel.
Also in U.S. patent No. 6630457 described modified hyaluronic acid produced by the reaction of primary amine with hyaluronic acid activated by a carbodiimide, such as EDC, and derived N-hydroxymethylamino, such as the NHS, at a pH of from 7.0 to 8.5. The compound obtained can be cross stitched in physiological conditions, such as glutaraldehyde, to obtain a hydrogel, which remains sensitive to glycosidases and almost completely degraded in less than 50 hours. The kinetics of this decomposition is compatible with the considered use as a vector for cells and growth factors, but is not suitable for use as filler, for example, in aesthetic surgery.
In conclusion, in the application WO 2006/021644 described a method of manufacturing poperechnogo hyaluronic acid by activation of hyaluronic acid cross-linking agent such as EDC, and a catalyst, such as the NHS, and subsequent reaction with a polypeptide, such as diligin, at a pH of from 4 to 10, PR is doctitle from 4 to 6. The pH may be raised at the end of the reaction to a value from 6 to 7 to enhance the yield of the extraction phase deposition. Thus, the cross-linking is carried out or in an acidic environment, which is then optionally neutralized or alkaline environment without subsequent modification of pH.
The applicant found that the use of acidic pH on the phase response is not always good for the amidation reaction and can, as mentioned above, lead to parasitic reactions, particularly reactions intramolecular esterification, can affect the physico-chemical properties of the resulting product.
Therefore, there remains a need to offer Poperechnaya hyaluronic acid, which can be obtained in dry form and then easily re-obtained in aqueous medium to form a hydrogel with suitable physicochemical properties, expressed, in particular, the elastic modulus G and the loss angle Delta is less than 30, and specified the hydrogel may be subjected to heat treatment, in particular sterilization, for use in the manufacture of the implant, is quite stable with respect to various factors of decomposition, such as enzymatic and/or bacterial exposure, temperature and free radicals, in order to fully rezorbiruetsa in vivo is less than for 4 months.
Zaya is ITIL absolutely accidentally discovered, what is the pH of the deposition of hyaluronic acid, poperechnogo with the polypeptide, in an organic solvent determines its rheological properties and sensitivity to the factors of the decomposition, such as temperature, free radicals and enzymes, such as hyaluronidase. After many experiments, the applicant has identified the optimum deposition conditions for obtaining poperechnogo hyaluronic acid, relatively insensitive to thermal decomposition, i.e. keeping their rheological properties after re-dissolution of precipitated compounds and sterilization. Is as if Poperechnaya hyaluronic acid, after re-obtain, retain a "memory" of its molecular organization at the time of deposition. Moreover, it was demonstrated that this molecular arrangement also affects the ability of the polymer to re-dissolve.
Without the need of binding theory, it is believed that the above process provides an opportunity to thicken and to be cured of the macromolecular network of hyaluronic acid not only through covalent bonds with the cross-linking agent, but also by ionic interactions and/or hydrogen bonds that develop during deposition.
The aim of the present invention is therefore Poperechnaya hyaluronic acid, which can be obtained by way which includes:
- activation of hyaluronic acid using a cross-linking agent and an auxiliary cross-linking agent for activated hyaluronic acid;
reaction of the activated hyaluronic acid with a crosslinking agent containing at least 50 wt.% the oligopeptides or polypeptide, in the environment of the reaction, adjusted to pH 8 to 12, to obtain Poperechnaya hyaluronic acid;
- regulation of the pH of a reaction medium to a value from 5 to 7;
- deposition poperechnogo hyaluronic acid in an organic solvent to obtain fibers poperechnogo hyaluronic acid and,
- optionally, drying the thus obtained fibers poperechnogo hyaluronic acid.
Poperechnaya hyaluronic acid obtained according to the invention is water-soluble. This expression is intended to mean that 1 g mentioned dehydrogenated fibers obtained as described above, broken down in several minutes and is completely dissolved in one liter of saline solution after a few hours without stirring.
Hyaluronic acid used in this method is usually used in its natural state, i.e. the state, naturally present in a living organism or dedicated bacteria in the production of bacterial fermentation. About what a rule it has a molecular weight of from 500,000 to 7000000 Dalton and is usually used in the form of sodium salt.
Hyaluronic acid activate before crosslinking using a crosslinking agent and an auxiliary crosslinking agent.
Examples of crosslinking agents are water-soluble carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-(3-trimethylaminoethyl)carbodiimide (ETC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC)and their salts and mixtures. In the present invention preferably use EDC.
Examples of auxiliary cross-linking agents are N-hydroxysuccinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazol (HOOBt), 1-hydroxy-7-asobancaria (HAt) and N-hydroxysultaine (sulfo-NHS), and mixtures thereof. Without limitation on the choice of the NHS in the present invention preferably use the latest.
The role of the agent and the auxiliary cross-linking agent is illustrated in Example 1 below.
According to the invention the molar ratio between the cross-linking agent and the links of the carboxylic acid hyaluronic acid is preferably from 2% to 200%, more preferably from 5% to 100%.
In addition, the molar ratio between the auxiliary cross-linking agent and cross-linking agent is preferably from 1:1 to 3:1, more preferably from 1.5:1 to 2.5:1, inclusive, and most preferably is 2.
Response to activation of hyaluronic acid with ivyshim agent can be carried out at pH for example, from 3 to 6, preferably from 4 to 5.
The concentration of hyaluronic acid in the reaction is, for example, from 0.1 to 5 wt.%, for example, from 0.1 to 1 wt.% inclusive.
A crosslinking agent contains at least 50 wt.% and preferably consists of oligopeptides or polypeptide, which may be random, block copolymer, segmented, graft, or star-shaped Homo - or copolypeptides. Cross-linking agent is usually salt, in particular the hydrochloride or the hydrobromide or especially triptorelin.
Examples of polypeptides that are suitable for use in the present invention are Homo - and copolymers of lysine, histidine and/or arginine, in particular polylysine having at least two or even at least five chains of lysine, such as, for example, diligin, polyhistidine and polyalanine. These amino acids can be in the form of D and/or L. For use in the present invention preferred diligin and its salts and derivatives.
According to the invention the number of functional amino groups of the polypeptide ranges from 1% to 100%, preferably from 10% to 50% of the number of functional groups of carboxylic acids used hyaluronic acid.
In the first preferred variant of the invention, the crosslinking agent is used in stoichiometric Koli is estwe about the functional amino groups of a crosslinking agent. Thus, at the end of the first step of the method according to the invention the number of activated functional groups carboxylic acid hyaluronic acid is equal to the number of functional amino groups, which will be added at the second stage.
In the second variant of the invention, the crosslinking agent is used in stoichiometric amount relative to the functional groups of the carboxylic acid hyaluronic acid. In this case, at the end of the first step of the method according to the invention the functional group of carboxylic acids of hyaluronic acid are activated, and the amount of crosslinking agent used in the second stage may, for example, be less than 30%, preferably less than 10%, or even about 5% (by number of moles of crosslinking agent relative to the number of moles of the functional groups of carboxylic acids).
The crosslinking reaction is usually carried out at a temperature and duration that is fully understandable to the expert, for example at a temperature 0-45°C, preferably 5-25°C for 1-10 hours, preferably 1-6 hours. In order to facilitate the formation of amide bonds, the pH of the reaction ranges from 8 to 12, preferably from 8 to 10 (inclusive). This pH value can be adjusted using any base, preferably a weak nucleophilic base, such as Diisopropylamine the (DIEA).
This reaction is usually conducted in a solvent, for example in an aqueous solution of sodium chloride.
The concentration of hyaluronic acid in the reaction is, for example, from 0.01 to 5 wt.%, for example, from 0.1 to 1 wt.% inclusive.
After the reaction the pH of a reaction medium is adjusted to a value from 5 to 7, preferably 5.5 to 7, using any acid, for example hydrochloric, before the deposition poperechnogo hyaluronic acid. The phase deposition is carried out in an organic solvent, such as ethanol, isopropanol, simple ether or acetone or mixtures thereof, and in this invention the preferred ethanol. The solvent is preferably used in excess of the amount of a reaction medium is 5 to 20 times, for example, approximately 10 times.
Then optionally carried out stage of drying in order to obtain digidrirovannoe form poperechnogo hyaluronic acid, which is easier to handle and which can be stored more conveniently. Storage can be carried out in conditions of negative temperatures.
The subject invention is also a method of manufacturing poperechnogo hyaluronic acid, which is described above.
This method can also include other stages than the above, and, in particular, the step of mixing the aforementioned dehydrated poperechnogo hyaluronic acid with a water solvent, for example, the R solution of sodium chloride, saline or buffer solution for injection (in particular, phosphate buffer saline)to form a hydrogel. The concentration of hyaluronic acid in the hydrogel can be from 1 to 4 wt.% and preferably from 1.5 to 3 wt.%/volume.
Therefore, the aim of the invention is also such a hydrogel containing Poperechnaya hyaluronic acid, in an aqueous solvent.
Thus obtained hydrogel is after sterilization, such as at 118 to 130°C for 2 to 30 minutes, according to the invention the elastic modulus G' of at least 100, for example in the range from 200 to 600 PA, inclusive, and the change in the elastic modulus less than 30%, preferably less than 20%, after heating at 93°C for 1 hour. He also preferably has a modulus of viscosity G" from 50 to 200 PA; the loss angle δ[=Inv tan(G"/G')] from 15 to 35° and a viscosity η of from 1000 to 3000 PA·C. Measurement of modulus of elasticity, modulus of viscosity and the loss angle can be carried out as follows: use the viscometer with cone and plate 4 cm, 4°, at 25°C. the Hydrogel is subjected to non-destructive testing viscosity-elasticity at 1 Hz with an applied strain of 1%. Measurement of modulus of elasticity is carried out using a rheometer AR 1000 company TA Instruments. The same device can be used to measure viscosity using a gradient shift 5×10-2c-1.
The purpose of the image is etenia is also sterilized hydrogel, containing hyaluronic acid, Poperechnaya cross-linking agent containing at least 50 wt.% the oligopeptides or polypeptide, characterized in that it has a change in the module of elasticity less than 30% after heating at 93°C for 1 hour.
This hydrogel is preferably used for the production of implants.
Such implants may, in particular, to be injected subcutaneously or intradermally into the fibrous tissue.
They may contain, in addition to the aforementioned hydrogel, vector liquid containing at least one polysaccharide, for example, at least one derivative of cellulose, such as carboxymethylcellulose, and/or at least one of glycosaminoglycan, such as sodium hyaluronate and/or particles are biocompatible, bioresorbable material, such as polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic) acid (PLGA), tricalcium phosphate (TCP) or hydroxyapatite (NAR) and their mixtures.
Examples of such materials for implants containing them, are described in particular in application WO 2004/069090.
The implants according to the invention are bioresorbability in the sense that they are ways to break down in the body for 6-18 months.
They can be in particular used for:
- deficiency of hyaluronic acid in the cavity or organ (usually d is rmacology, aesthetic medicine or orthopedic operations);
restore volume that has elapsed during surgical interventions (usually in eye surgery) or
- application on top of the normal or damaged dermis (usually in cosmetology and dermatology).
The above implant is particularly suitable for use in filling facial wrinkles and fine lines and/or scars on the body.
Therefore, the aim of the present invention is also the use of poperechnogo hyaluronic acid, which is described above, for the production of injectable implants for use in aesthetic and/or cosmetic surgery or for the production of fillers, in particular products for filling wrinkles, fine wrinkles, scars or depressions on the skin, for example, with lipodystrophy.
Now the invention will be illustrated in the following non-restrictive examples.
Example 1: Synthesis of hyaluronic acid, poperechnogo the polypeptide according to the invention
1. The reaction scheme
The following reaction scheme may be illustrated as follows (example taken diligin):
The crosslinking reaction (scheme 1) consists of a dual education peptide bonds between the functional groups of the two chains of hyaluronic acid and funk is optional amino dilysine. As cross-linking reagents using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
The mechanism of the crosslinking reaction can be illustrated as follows:
The first stage consists of a nucleophilic effects of functional group carboxylic acid hyaluronic acid carbodiimide functional group cross-linking agent EDC. The resulting O-allocating then replaced by the NHS for the formation of more stable activated complex ether (product 1-ethyl-3-(3-dimethylaminopropyl)urea). In fact, 0-allocating can be converted into inert N-allocatio in weakly acidic aqueous medium and long reaction time. The last step is the nucleophilic impact of one of the functional amino groups dilysine (preferably finite space favorable) on the activated ester to form an amide bond, liberating the NHS.
1st stage: the stage of swelling
3 g of sodium chloride successively added to 300 ml of milliQ water in a glass reactor with a volume of 500 ml After dissolution of sodium chloride in sonicator 2 g of hyaluronic acid (HTL Sarl, batch No. 1016 PH, Mw=2.6×106Daltons, called below "ON") introduced into the reactor containing saline, thoroughly and the best possible is about separating the fibers by hand. After stirring heterogeneous environment with a spatula for 1 minute, the reactor was left at 4°C for 15 hours without stirring and closed with aluminum foil to protect the reaction environment.
2nd stage: stage stitching
The reaction mixture was removed from the refrigerator and was stirred at ambient temperature (18-25°C) for 10 minutes (visual solution should be fully transparent and homogeneous, slightly viscous, like liquid honey).
The mechanical stirring using a Teflon stir bar in the shape of a Crescent. Speed was 60 rpm
Next, a solution of 464 mg (a 4.03 mmol) of N-hydroxysuccinimide (ACROS, 98%purity, called below "NHS") in 5 ml of milliQ water was prepared in a test tube for hemolysis and shook in a circular motion to dissolve the whole of the NHS. This solution was added drip to the environment of the reaction at a speed of 5 ml/min
This mixture was left to mix for 5 minutes, then 313 mg (2.02 mmol) of a solution of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (Sigma-Aldrich, No. 03450-5G, called below "EDC") was added in 4 ml of milliQ water. Dissolution was performed by shaking in a circular motion and then added infusion at a rate of 5 ml/min
The mixture was left to mix for 30 minutes and then an aqueous solution dilysine was added to the reaction environment at a rate of 1 ml/min, This solution was prepared by restorani is in 1 ml milliQ water, when shaken in a circular motion, 233 mg (0.67 mmol) of the hydrochloride dilysine (provider VASHEM, No. G2675) and then 1302 ál (10,08 mmol) diisopropylethylamine (provider ACROS No. 115225000, called below "DIEA"), all in vitro hemodialysis. This blend is two different phases of forming a reversible emulsion after intensive mixing. An attempt was made to mix the maximum amount of the emulsion by adding it to the reaction environment. The pH of the reaction environment should be between 8.5 to 10.5.
All left to mix for 3 hours.
3rd stage: stage cleaning
After stopping stirring the pH of the solution was adjusted to deposition using 1M HCl to reduce the pH to 5.7.
Then I prepared the reactor volume of 1 liter with a mechanical stirrer and stirring rod in the form of a comb. 420 ml of 95° ethanol was poured into the reactor and included mechanical stirring at a very high speed (about 1000 rpm).
42 ml of reaction mixture containing poperechnyy hyaluronate, then selected using a syringe with a capacity of 50 ml and was then introduced continuously as a thin layer in the reactor. The solution should be clear, colorless and quite viscous.
Immediately after introduction of the mixing continued for another two minutes. Then the stirring rod was removed from the reactor and the polymer was decomposed n is the Frit with a porosity II, using a pair of tweezers. The polymer was quickly dried in a vacuum flask to a maximum of 15 seconds and was left to dasyati in an oven under vacuum for a period of not less than 12 hours. The final product should be pure white.
4th stage: the stage of re-manufacturing
For the manufacture of 10 ml of gel with a concentration of 2.4%, 240 mg of dried Poperechnaya polymer introduced in standard polypropylene syringe with cap (on the exit hole). Then the solid material was added to 10 ml of buffer** solution and all left to swell at 4°C for 12-15 hours.
After removal of the syringe from the refrigerator, the product was quickly mixed using a mechanical stirrer at 1000 rpm as the stirring rod used laboratory spatula stainless steel in the form of a spoon. For this product mixing time was approximately 5 minutes, but it can vary depending on the viscosity. The final gel should be colorless and completely uniform.
Example 2: Check for decay or stability
Checking for rapid decomposition, which give information about the resistance of the polymer to various factors decomposition in vivo, conducts specialist (see, in particular, the document FR 2861734).
In this example, conducted one such review, which included the measurement of the rheological characteristics of the cross is Ossetic, pre-sterilized products, which are then subjected to heating at 93°C for one hour. Then calculate the loss elastic modulus (G') in percent after heating. The lower this percentage is, the more resistant to the effects of heat and how it is considered, it is more resistant to the effects of other factors decomposition. Therefore, this check is a prediction of the degree of decomposition in vivo poperechnogo hyaluronic acid and, therefore, the forecast duration of the filling of wrinkles, which can be achieved.
All tested products were sterile.
Several commercially available products were tested with:
Product 1, which was hyaluronic acid obtained according to Example 1, and
Product 2, which was hyaluronic acid obtained according to Example 1, except that used were 45 mol.% EDC; 90 mol.% NHS and 15 mol.% dilysine regarding the number of moles of links COOH hyaluronic acid and attitude 2,22 DIEA/NHS.
Table 1 shows the results obtained for the different tested Poperechnaya hyaluronic acid.
|Check for decomposition poperechnogo hyaluronan is howling acid|
|Sterile sample||T0||T1 after 1 hour at 93°C||% losses : G'|
|The elastic modulus (G')||The angle of losses||The elastic modulus (G')||The angle of losses|
|Synthesis parameters Poperechnaya hyaluronic acid|
|Product||% EDC||% dilysine||Attitude DIEA/NHS||the pH during the precipitation|
|Product 3||100%||200%||5%||2,0||the 5.7|
|* regarding the number of moles of functional groups carboxylic acid hyaluronic acid.|
Evaluated the physicochemical properties of the above products after re-manufacture, as described in Example 1, before and after one hour in an incubator at 90°C. More specifically, the estimated WASC is here hydrogel and measured its modulus of elasticity. The results are presented in table 3.
Used classes from 1 to 5, which represent a summary measure that takes into account the elasticity and the viscosity of the gel. What was considered to be more elastic gel, the higher the score. Conversely, heterogeneous and/or liquid gel has a lower score.
|Physico-chemical properties Poperechnaya hyaluronic acid|
|Product||The appearance of the hydrogel (t0)||G' (t0)||The appearance of the hydrogel (KZT60)||G' (KZT60)||% losses : G'|
|Product 1||Transparent, slightly grainy (Class 5)||262||Transparent, elastic (Class 5)||224||14%|
|Product a||Almost viscoelastic (Class 2)||-||-||-||-|
|Product||Transparent, viscous (Class 4)||-||Transparent (Class 3)||-||-|
|Product 3||Very transparent (Class 5)||296||Very transparent (Class 5)||215||27%|
|Product C||Very transparent (Class 5)||-||Very transparent (Class 2)||-||-|
|Product 4||Transparent, slightly grainy (Class 5)||206||Transparent, elastic (Class 5)||199||%|
|Product D||Very transparent, viscoelastic (Class 5)||-||Almost viscoelastic (Class 2)||-||-|
The table shows that Poperechnaya hyaluronic acid, precipitated under alkaline pH, but could be easily re-obtained as hydrogels, do not give hydrogels suitable for the product used for the filling up wrinkles. It is assumed that this phenomenon is caused by the insufficient development of ionic bonds during deposition.
In addition, Poperechnaya hyaluronic acid, precipitated with overly acidic pH, give hydrogels with good viscoelasticity (provided that they can be re-made, which is not always possible), but which explicitly decompose when placed in the incubator and will therefore be sensitive to endogenous factors of decomposition.
In fact, it seems that only the pH values of precipitation from 5 to 7 give the opportunity to easily obtain a homogeneous hydrogel with very satisfactory viscoelasticity, which is not reduced significantly after checking on decomposition. This confirms that in this pH range the macromolecular network, formed electrostatic and covalent bonds, is optimal for use as a filler.
1. The method of obtaining poperechnogo hyaluronic acid, containing:
- activation of hyaluronic acid using a cross-linking agent and an auxiliary cross-linking agent for activated hyaluronic acid;
reaction of the activated hyaluronic acid with a nucleophilic cross-linking agent containing at least 50 wt.% the oligopeptides or polypeptide, in the environment of the reaction, adjusted to pH 8 to 12, to receive the cross is assetou hyaluronic acid;
- regulation of the pH of a reaction medium to a value from 5 to 7;
- deposition poperechnogo hyaluronic acid in an organic solvent to obtain fibers poperechnogo hyaluronic acid.
2. The method according to claim 1, characterized in that the crosslinking agent is chosen from: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 1-ethyl-3-(3-trimethylaminoethyl)carbodiimide (ETC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC)and their salts and mixtures.
3. The method according to claim 1 or 2, characterized in that the auxiliary crosslinking agent selected from: N-hydroxysuccinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-isobenzofuranone (HAt) and N-hydroxymaleimide (sulfo-NHS) and mixtures thereof.
4. The method according to claim 1 or 2, characterized in that the molar ratio of crosslinking agent with links carboxylic acid hyaluronic acid is from 5% to 100% inclusive.
5. The method according to claim 1 or 2, characterized in that the molar ratio of the auxiliary cross-linking agent cross-linking agent is from 1:1 to 3:1, inclusive.
6. The method according to claim 1 or 2, characterized in that the reaction of the activated hyaluronic acid cross-linking agent is carried out at a pH of from 3 to 6.
7. The method according to claim 1 or 2, wherein the polypeptide is a Homo - or copolymer of lysine.
8. The method according to claim 7, characterized those who, that homopolymers lysine is diligin.
9. The method according to claim 1 or 2, characterized in that the crosslinking agent is used in stoichiometric amount relative to the functional amino groups of a crosslinking agent.
10. The method according to claim 1 or 2, characterized in that the crosslinking agent is used in stoichiometric amount relative to the functional carboxyl groups of hyaluronic acid.
11. The method according to claim 10, characterized in that the amount of crosslinking agent used in the second stage, less than 30% of the number of moles of crosslinking agent relative to the number of moles of the carboxyl functional groups.
12. The method according to claim 1 or 2, characterized in that the crosslinking reaction is carried out at a pH from 8 to 10.
13. The method according to claim 1 or 2, characterized in that the pH of precipitation is from 5 to 7.
14. The method according to claim 1 or 2, characterized in that the organic solvent is ethanol or isopropanol.
15. The method according to claim 1, characterized in that the method further comprises drying the obtained fibers poperechnogo hyaluronic acid.
16. Poperechnaya hyaluronic acid obtained by the method according to claim 1 or 2.
17. The hydrogel, characterized in that it contains Poperechnaya hyaluronic acid for P16 in the buffer aqueous solvent.
18. Use poperechnogo hyaluronic acid in article 16, for the production of injectable the implants for use in aesthetic and/or cosmetic surgery or for the production of fillers for filling wrinkles, small folds, scars or depressions on the skin.
SUBSTANCE: disclosed is a method of determining antibacterial properties of chitosan by estimating its minimum bacteriostatic and/or bactericidal concentration. Complex buffer solutions based on three organic acids MES, ACES and TES with different pH values are prepared. The ready buffer solutions are poured into a vessel. Double dilutions of chitosan are then prepared in vessels with the buffer solutions. Aliquots of a bacterial suspension in a fluid medium are added to the chitosan solutions in the buffer. The solutions are incubated for 24 hours at temperature which is optimum for bacterial growth. The minimum bacteriostatic and/or minimum bactericidal concentration of chitosan is then determined after incubation by determining growth of the culture or a drop in the number of living cells, respectively.
EFFECT: invention enables to determine antibacterial properties of chitosan in a wide pH range from 5,50 to 8,00 without the need to use buffers of different chemical composition.
5 dwg, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to a method for preparing sodium salt of hyaluronic acid modified by boron compounds with no fluid medium added. The method consists in the fact that powdered sodium salt of hyaluronic acid together with a modifying agent and mixed modifying agents is pre-homogenised in a mixer at temperature ranging within 20° to 50°C; thereafter the prepared homogenous powder mixture is simultaneously exposed to pressure and shearing deformation in a mechanochemical reactor at temperature ranging within 20° to 50°C and pressure 5-1000 MPa.
EFFECT: invention provides preparing boron-containing sodium salt of hyaluronic acid applied in boron neutron capture therapy in one-stage process parameters with no fluid medium added which requires low power, labour and water consumptions.
13 cl, 15 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to medicine, more specifically to producing chitosan oligomers possessing biological activity and applicable in food industry and medicine. In a method for producing chitosan oligomers, a chitosan solution is taken in the concentration of 0.025-0.075% (weight/volume) and exposed to low-frequency ultrasound of the intensity of 92-460 Wt/cm2 for 5-30 minutes.
EFFECT: reduction in price of the chitosan oligomers production combined with promotion of higher medium viscosity molecular weight of the product within the range 25 ÷ 120 kDa.
3 tbl, 3 ex
SUBSTANCE: method involves preliminary acetylation of chitin with acetic anhydride, washing and drying the acetylated chitin in order to reduce degree of deacetylation thereof and, as a result, increase output of the desired product - D(+)-glucosamine hydrochloride when obtaining said product through hydrolysis of acetylated chitin with concentrated hydrochloric acid while heating, followed by evaporation, crystallisation, separation, washing and drying the desired product.
EFFECT: high output of the desired product while maintaining its high quality; method is more environmentally friendly since pre-treatment of chitin reduces the amount of processing wastes.
1 cl, 2 ex
SUBSTANCE: method of producing chitosan chromate involves reaction of soluble chitosan salts with metal chromates in ratio of 2 moles of the chitosan cation to 1 mole of chromate anion or with metal bichromates in ratio of 4 moles of the chitosan cation to 1 mole of the bichromate anion. The solid chitosan chromate residue formed is then separated and dried at temperature not higher than 150°C. The invention discloses an energy-intensive composition based on chitosan dodecahydro-closo-dodecaborate containing an effective amount of chitosan chromate. The quantitative ratio in the energy-intensive composition is by the required combustion mode: the higher the content of chitosan chromate, the higher the activity of the composition.
EFFECT: invention enables to obtain a chemical compound having sufficiently high oxidative properties and suitable for use in energy-intensive compositions which burn without emitting harmful gaseous products.
3 cl, 5 ex
SUBSTANCE: method involves taking a certain weighed amount of chitosanium chromate which is first purified from extraneous impurities and reduced to constant weight. The weighed amount is then turned into a stable weighted form through thermal treatment on air at temperature 800-900°C to form chromium oxide Cr2O3. The weight of the formed chromium oxide is then determined. Content of chromic acid in the initial weighed amount of chitosanium chromate is then calculated from the weight of chromium oxide. The degree of deacetylation of chitosan is calculated using defined formulae.
EFFECT: invention enables to increase accuracy of determining degree of deacetylation of chitosan.
SUBSTANCE: invention relates to a method of extracting and stabilising ultra low-molecular aminoglycans from eggshell wastes. Aminoglycan extract is used to produce cosmetic creams with skin moisturising and anti-wrinkle properties. The method of extracting low-molecular aminoglycan compound of formula I from a natural source of eggshell wastes, which consists of alternating glucuronic acid and N-acetylglucosamine units, where M can be one or more of Na, Ca, K, Mg; and n is a whole number from 20 to 40, involves the following steps: (a) preparing eggshell wastes for extraction of embryonic low-molecular aminoglycan compound of formula I using a polar organic solvent in water, (b) extracting low-molecular aminoglycan compound of formula I in form of a water-soluble salt, for which the eggshell from step (a) is vigorously shaken with aqueous polar salt solution at 10°C - 35°C for 6-12 hours, then filtered or centrifuged in order to collect an aqueous layer containing a dissolved aminoglycan compound of formula I; (c) extracting a purified low-molecular aminoglycan compound of formula I by forming a gel from an aqueous mixture of salts using a polar organic solvent, for which the solution from step (b) is successively and step-by-step mixed with an organic solvent mixed with water while gently stirring and then cooled to maintain temperature from 20°C to 25°C, and the formed gel is left for 2-24 hours for complete precipitation, then filtered or centrifuged in order to extract a semidry aminoglycan compound of formula I; (d) the extracted aminoglycan compound of formula I from step (c) is stabilised via gradual addition of organic oils to the semidry gel to form aminoglycan compound of formula I. In order to prepare a composition having anti-wrinkle properties, at least one pharmaceutically acceptable filler is added to the stabilised aminoglycan compound of formula I obtained at step (d).
EFFECT: method enables to obtain an aminoglycan compound of formula I with the necessary viscosity and skin penetrating properties for reducing skin wrinkles, as well as excellent softening and moisturising effects.
8 cl, 9 ex
SUBSTANCE: method involves feeding wastes to be treated into artificial containers, biotreatement, tapping the filtrate and removing the obtained biomass. Biotreatement is carried out by culturing hoverfly larvae from the freshly laid eggs phase to the pupation phase in the fermented wastes to be treated, placed in artificial meshed containers the bottom and walls of which are covered with filter cloth. The apparatus has artificial containers, devices for feeding the wastes to be treated, outputting the filtrate and collecting the biomass. The artificial containers have a meshed bottom and walls covered with filter cloth.
EFFECT: invention enables to combine biotreatment of methane wastes with production of chitin containing biomass.
8 cl, 1 dwg
SUBSTANCE: method of producing nanoparticles of low-molecular chitosan involves preparing a solution of pre-purified low-molecular chitosan in filtered 1-2 wt % aqueous acetic acid, adding solutions of hydroxides of alkali metals or ammonia for 2 hours, dispersing the system using a mechanical mixer at a rate of 200-300 rpm at temperature 20°C to pH 6.9-7.0. Further, the dispersion is centrifuged at 10000 rpm. The obtained solid residue is redispersed in bidistillate while mechanically mixing at a rate of 200-300 rpm at temperature 20°C.
EFFECT: invention simplifies the method of producing nanoparticles of low-molecular chitosan and apparatus design without formation of undesirable harmful substances.
SUBSTANCE: starting high-molecular chitosan is dissolved in acid solution. The chitosan dissolved in the acid is then precipitated by adding alkali solution. The re-precipitated high-molecular chitosan is washed from the formed salt and excess alkali using a coarse-porous filter. The re-precipitated chitosan is dissolved in acid solution until achieving pH 5.5. An enzyme preparation is then added and hydrolysis is carried out. The reaction is stopped after formation of low-molecular chitosan.
EFFECT: method is characterised by avoiding the need to remove salts from the enzymatic mixture and the end product, as well as low level of loss of material.
2 dwg, 7 tbl, 6 ex
FIELD: fish industry.
SUBSTANCE: method involves providing deacetylation of raw material with the use of preliminarily cooled alkaline solution; washing and drying. Deacetylation process is performed in three stages, first stage being performed for 7 days and subsequent two stages being performed for 2 hours each, combined with thermal processing at temperature of 55-590C. Washing process is provided after each deacetylation stage.
EFFECT: provision for producing of chitosan from chitin of cancerous with increased extent of deacetylation, while native properties of natural polymer being kept, without breaking of glycoside binding chain.
FIELD: organic chemistry.
SUBSTANCE: claimed method includes subsequent chitosane-containing raw material with non-polar liquefied gas, water, alkali, water, acid, water, alkali, and water to produce target product in form of solid residue, wherein in at least first extraction step pressure in reaction mixture is periodically released to provide extractant boiling, and than increased up to starting value.
EFFECT: method with reduced energy consumption.
FIELD: chemical technology of natural compounds.
SUBSTANCE: invention describes a method for preparing water-soluble derivatives of chitosan. Method involves treatment of chitosan with acid medium up to its swelling wherein vapor medium water-acid is used as acid medium. Treatment of chitosan is carried out with vapor of monobasic acid aqueous solution taken among the group including hydrochloric acid, formic acid and acetic acid. Method allows simplifying technology in preparing water-soluble derivatives of chitosan.
EFFECT: improved preparing method.
4 cl, 1 tbl, 9 ex
FIELD: chemistry and technology of derivatives of polysaccharides, chemical technology.
SUBSTANCE: invention relates to methods for preparing chitosan esters. Invention describes a method for preparing chitosan polyethylene glycol ester that involves dissolving chitosan in acetic acid followed by alkalization. Then the reaction mixture is subjected for effect of ethylene oxide under pressure 1-3 atm and temperature 60-100°C, and the concentration of reaction mass is corrected by addition of distilled water up to the density value of solution 1.030-1.032 g/cm3. Then the reaction mass is purified by electrodialysis at the rate value of solution in treatment chambers 3.0 cm/s, not less, temperature 20-45°C, the current density value 0.25-0.75 A/dm2 and the constant volume of the reaction mass. Method provides enhancing the effectiveness of purification by electrodialysis due to reducing energy consumptions. Chitosan esters can be used in medicine, cosmetics, food and chemical industry.
EFFECT: improved preparing method.
FIELD: organic chemistry of natural compounds, chemical technology, medicine.
SUBSTANCE: invention relates to the group of chitosan-containing compounds. Invention relates to synthesis of modified chitosan of the following structure: wherein n = 150-1400. The modified chitosan possesses the bactericidal activity, in particular, antituberculosis activity.
EFFECT: valuable medicinal properties of modified chitosan.
1 tbl, 1 dwg, 3 ex
FIELD: natural compounds technology.
SUBSTANCE: chitosan preparation process comprises breaking naturally occurring chitin-containing material, charging it into reactor, demineralization with 6-7% aqueous hydrochloric acid, deproteination with sodium hydroxide solution at 85-95°C, deacetylation with sodium hydroxide solution on heating, decoloration, and washing with water after each stage to pH 6.5. Process is characterized by that chitin-containing material broken to achieve fraction 0.5-6 mm is fed simultaneously into a number of reactors, wherein demineralization is effected with aqueous hydrochloric acid stream at 85-95°C for 1.5 h while controlling pH in each reactor exit to achieve acid concentration in each reactor exit the same as concentration of the initial acid by way of feeding it in a continuous manner. In addition, deproteination is carried out with 6-7% sodium hydroxide solution stream for 1.5 h followed by discharging treated material into autoclave to perform deacetylation simultaneously with decoloration using 50% sodium hydroxide solution at 130-140°C in inert gas environment and in presence of 3-5% hydrogen peroxide solution used in amount 3-5% of the total volume of mixture.
EFFECT: enhanced process efficiency.
FIELD: chemical technology.
SUBSTANCE: invention relates to methods for preparing water-soluble saline complexes (associates) of hyaluronic acid with d-metals of IV, V and VI periods of Mendeleyev's periodic system of elements that can be used in pharmacology and cosmetology. Invention describes a method for preparing water-soluble saline complexes of hyaluronic acid involving preparing an aqueous solution of salt of d-metal of IV, V and VI periods of periodic system and its mixing with hyaluronic acid sodium salt, holding the mixture, its stirring, dilution with water and isolation of the end product. For mixing method involves using the amount of aqueous salt of abovementioned d-metal that is equivalent to the amount of carboxy-groups of hyaluronic acid sodium salt or in the limit from 0.95 to 1.10. After dilution with water the solution mixture is subjected for ultrafiltration on separating membranes with simultaneous washing out with aqueous salt solution of abovementioned d-metal firstly and then with deionized water followed by concentrating the product. By another variant for mixing the method involves the amount of aqueous solution of d-metal salt lesser of the equivalent amount of carboxy-groups in hyaluronic acid sodium salt. After dilution with water the mixture is subjected for ultrafiltration on separating membranes with simultaneous washing out with deionized water followed by concentrating the product also. Method is characterized by the decreased time of processes and simplicity.
EFFECT: improved preparing method.
2 cl, 1 tbl
FIELD: medicine, food processing industry, in particular production of depolymerized chitosane and products based on the same.
SUBSTANCE: claimed method is based on using of chitosanase in acetic acid medium and spray drying of and depolymerized chitosane and is characterized in that obtained depolymerized chitosane is preliminary converted in non-ionized form by neutralizing of bound acetic acid with ammonium hydroxide followed by precipitation in ethanol and air drying. Further interaction is carried out with ammonium lipoate or glutathione in aqueous medium. Claimed products may be used individually or in combination with other components.
EFFECT: new products for food processing industry and medicine.
4 cl, 2 ex
FIELD: organic chemistry, chemical technology.
SUBSTANCE: invention relates to a method for preparing modified glycosaminoglycans possessing analgesic properties. Method involves interaction of glycosaminoglycans with 1-phenyl-2,3-dimethyl-4-aminopyrazolone-5-(4-aminoantipyrine) in aqueous medium at pH = 4.7-4.8 in the presence water-soluble 1-ethyl-3-[3-(dimethlamino)propyl]carbodiimide as a condensing agent at room temperature followed by purification from low-molecular reagents. Method involves a single step that simplifies technology in preparing modified glycosaminoglycans.
EFFECT: improved preparing method.
FIELD: natural substances, chemical technology.
SUBSTANCE: invention relates to a method for preparing chitosan and purification from components of the reaction mixture - low-molecular products of deacetylation and alkali excess. Invention relates to a method for purifying chitosan prepared by solid-state method involving treatment of reaction mass with extractant consisting of 3.3-20.0% of water, 32.2-57.1% of ethyl acetate and 24.6-64.5% of ethanol at the extractant boiling point. Also, invention relates to a method for purifying chitosan prepared by suspension method and involving treatment of the reaction mass with ethyl acetate and the following treatment with extractant consisting of 6.2-25.0% of water, 12.5-62.5% of ethyl acetate and 31.3-62.5% of ethanol at the extractant boiling point.
EFFECT: improved isolating and preparing method.
3 cl, 2 tbl, 1 dwg