The method of obtaining a modified hyaluronic acid


(57) Abstract:

Describes how to obtain a chemically modified hyaluronic acid, which consists in the activation of carboxyl groups of hyaluronic acid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and reaction of the resulting O-allodapini with a water suspension of 5-aminosalicylic acid in the initial reaction temperature 40oC for 2-4 hours, followed by purification of the polymeric amide dialysis, aluminum oxide and precipitation with ethanol. Depending on the time of reaction get hyaluronic acid with different degree of substitution of carboxyl groups. Film of the modified polysaccharide can be used as a biomaterial in abdominal surgery to protect the anastomosis at the resections of the bowel, for impregnation of suture material (sutures), artificial limbs, etc., 1 table.

The invention relates to the field of biochemistry and can be used in medicine.

Hyaluronic acid (ha), a natural polysaccharide from the class of glycosaminoglycans, consists of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine (see formula I in the end of the description).

GK is michlet is obtained by using producing Ledger special bacteria.

Ledger is a polyanionic polymer with a molecular weight from several hundred Yes up to 10 million Yes (average M=1-2 million Yes). These characteristics determine the unique physico-chemical properties of the polysaccharide and its derivatives.

Biological function GK can be divided into "passive" and "active". As an inert material, hyaluronic acid is involved in tissue homeostasis, steric regulation (osmosis) penetration of any substance, acts as a "lubricant" that improves the mobility of joints, etc.

"Active" functions GK are the specific binding with proteins in the extracellular matrix and on the cell surface; this interaction plays an important role in the formation of cartilage tissue in the processes of cell proliferation, morphogenesis and embryonic development of animals, as well as in the mechanisms of inflammation and cancer.

Ledger is used as adjuvant in ophthalmology (drugs visk, Healon) as the matrix in the so-called "delivery systems" of drugs and surgery to prevent polaprezinc tissue adhesion (adhesive disease). It is established that the General Ledger increases the absorption of drugs and some proteins through the mucous membranes, and appolina, significantly improved in combination with the General Ledger. In combination with the group some of the drug substance form in the epidermis like depo these drugs and are released from the composition slower than in the absence of ha.

Functionalization of the Ledger or the synthesis bioconjugates with SC has a number of advantages compared with simple mixtures GK-medicine. First, chemical modification allows you to change the physico-chemical properties of ha under the appropriate application and may significantly affect the clearance derivatives of GA. Secondly, bioconjugate Ledger may have greater solubility compared with the actual medication. Thirdly, the hydrogels GK-medication can be used for local controlled release of substances in the target organs. And, finally, the high affinity receptor Ledger in metastasising cells can be used for targeted delivery of anticancer agents to target cells.

As you know, polymeric materials are widely used for manufacturing various medical devices. Synthetic polymers are optimized mechanical properties and often do not have sufficient biocompatibility; on the contrary, the biopolymers are usually not necessary meone to obtain composites with desired physico-chemical and biological properties.

Composition Ledger with polyacrylic acid is prepared by dissolving both polymers in water in different ratios, then form a film, which lyophilizer and subjected to a crosslinking reaction by heat treatment at 130oC in vacuum for 24 h [Cascone, M., Sim, C., Downes S. Blends of synthetic and natural polymers as drug delivery systems for gormone. // Biomaterials. - 1995. - V. 16. - P. 569]. Hydrogels GK-polyvinyl alcohol are also manufactured by dissolving both polymers in water in different proportions.

On the basis of a mixture of two anionic polymers: General Ledger and carboxymethyl cellulose (CMC) were created bioabsorbable film materials Seprafilm used to prevent postoperative adhesions. In the presence of water-soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (CBI) to the carboxyl groups of ha or CMC were attached Quaternary ammonium groups, which increased the interaction between the polymer chains of the components of the mixture [US Patent 5017229. - 1991; Burns J., Burgess L., Skinner, K. , Rose, R. et al. A hyaluronate-based gel for the prevention of postsurgical adhesion: Evaluation in two animal species. // Fertil. Steril. - 1996. - V. 66. - P. 814].

Known manufacturing techniques of composite material Ledger and collagen, which is based on the integration of both components in coagula composite materials and their modification by crosslinking. // J. Biomed. Mater. Res. - 1996. - V. 30. - P. 369]. Atelocollagen, suspended in 0.5 M CH3COOH at pH 3.2, and added to a solution of ha. After coagulation, washing and drying the precipitate was obtained a thin film, which is then subjected to the stitching in the solution of the dialdehyde.

Known polymeric complexes of GA and its sodium, potassium or lithium salts with natural polysaccharides and proteins: chitin, alginic acid, fibrin, collagen, etc. that can be applied as a coating to various medical devices, such as artificial vascular prostheses [EP 0544259 A1].

New biomaterials based on GK can be obtained by a variety of reactions according to the functional groups of the polymer. For example, when the esterification reaction Ledger at the first stage form tetraalkylammonium salt Ledger in DMF, followed by reaction with primary alcohols, for example ethyl, propyl, pencilbeam, benzyl or dodecylamine [Hant J., Joshi H., Stella V. J. et al. Diggusion and drug release in polymer films prepared from ester derivatives of hyaluronic acid. // J. Controlled Rel. - 1990. - V. 12. - P. 159] . Depending on the nature of alcohol and the number of modified groups GK can widely vary the properties of the obtained materials.

Using the reaction of esterifies xinnuo group. Partial esters of ha with anti-inflammatory steroids were synthesized by esterification reaction with the preliminary formation tetrabutylammonium CC salt. Fully or partially esterified with hydrocortisone SC is currently under investigation as a new biomaterial in various dosage forms, such as microspheres, tablets, pills or films [Benedetti L. M., Torr E. M., Stella V. J. Microspheres of hyaluronic acid esters - fabrication methods and in vitro hydrocortisone release. // J. Controlled Rel. - 1990. - V. 13. - R. 33].

Using the method of carbodiimide activation synthesized many bioconjugates Ledger with drugs. Most of the conducted experiments based on the reaction of the OED-activated carboxyl of D-glucuronic acid in the group with a compound containing amino groups.

Ester of chloramphenicol with alanine was attached to various glycosaminoglycans through CBI-activated carboxypropyl polysaccharides and amino group of alanine. Similarly amino anthracycline antibiotic adriamycin (doxorubicin and daunomycin, and mitomycin were associated with active groups GK [Sega S., M. Terbojevich et al. Antracycline antibiotics supported on water-soluble polysaccharides: Synthesis and physicochemical characterization. the genoway acid and others, one part of carboxyl groups which is used for cross-linking of the macromolecules, the other part tarifitsiruetsya mono - or polyalcohol or interacts with amines, including various medicinal substances. The activating agent carboxypropyl in this case is 2-chloro-1-methylpyridinium, and reaction with alcohols or amines is conducted by heating or exposure to radiation [EP 341745] . The materials synthesized using the described method, can be used for the manufacture of pharmaceutical compositions, various medical devices such as surgical sutures, films for the regeneration of the epidermis, for the treatment of skin diseases, etc.

To reduce the solubility of the civil code and, therefore, prolongation of the action of medicines used it in combination, a crosslinking reaction Ledger carboxyl or hydroxyl groups of the polymer. Cross-linking reagents can be oxychloride phosphorus (V), diphenylsulfone, epoxysilane, dialdehyde, diamines, dihydrazide, etc. In some reactions intra - and intermolecular crosslinking of polymer chains Ledger, for example, using dihydrazide adipic acid requires the use of the OED [Vercruysse, K. P., Prestwich G. D. Hyauronate derivatives in drug"ptx2">

The reaction of the so-called ionic crosslinking Ledger performed using ferric salts: get the hydrogel SC with 0.5% FeCl3(company Ethicon) [Johns D. B. , Rodgers, K. E., Donahue W. D. et al. Reduction of adhesion formation by postoperative administration of ionically cross-linked hyaluronic acid. // Fertil Steril. -1997. - N 68(1). - P. 37-42]. This hydrogel is intended to prevent postoperative adhesions disease in abdominal surgery.

Closest to the proposed invention is the obtaining of the civil code, modified by carboxyl groups-aminocaproic acid. -Aminocaproic acid is known hemostatic agent. Connection-aminocaproic acid is carried out by activation of the acid groups of ha with the help of the OED or N-hydroxysuccinimide. In this case are derivatives of hyaluronic acid, soluble in organic solvents. Secondarily activating using the OED already carboxyl group-aminocaproic acid can be further modified Ledger, for example, daunomycin [EP 0506976 B1, 1992].

The authors chemically modified GC obtained from GC and 5-aminosalicylic acid (5-ASA) method, carbodiimide activation of the carboxyl groups of the polysaccharide. A modified GA with RAE 1. 1 g GK dissolved in 50 ml of water with a pH of 4.7 and 4.8, add 0,476 g of water-soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. Then this solution under vigorous stirring poured to 0,382 g 5-ASA, suspended in 40 ml of water, with a pH of 4.7-4.8 at 40oC. After adding all of the solution the temperature of the reaction mixture was lowered to room temperature and the reaction is carried out for 1 hour. Upon completion of the reaction in order to remove low molecular weight starting compounds a solution of modified GC cialiswhat against acidified distilled water for 24 h, and then the product was then purified on a column of aluminum oxide. After purification on a column of the pH of an aqueous solution of the polymer is brought Hcl to 3-4 and the precipitated 4-fold volume of ethanol, the precipitate centrifuged, washed with ethanol, then ether and dried in vacuum. So get a modified GA with the acid groups of 5-ASA with a degree of substitution of carboxyl groups of the polymer 30 mol.% (see diagram at the end of the description). The number of 5-ASA, covalently linked to the polymer, determined photometrically at 304 nm after hydrolysis exact sample of the product in 20% hydrochloric acid. Immediately after the deposition of the modified GK (acid) water soluble after wyer precipitated by alcohol from a solution with a pH of 8.0 to 8.5, you can obtain a sodium salt of a modified GC (see formula II in the end of the description). Sodium salt of a modified hyaluronic acid is water soluble polymer, which can produce drug films for various purposes.

Example 2. The reaction of GA with 5-ASA carried out analogously to example 1. The reaction time is 4 hours With the number of 5-ASA, covalently associated with the polymer is 50 mol.%.

Depending on the time of carrying out the reaction it is possible to obtain products with different degree of substitution of carboxyl groups of ha.

As all salicylates, modified GC capable of forming colored complexes with Fl3that are poorly soluble in water connections. Processing the surface of the films of the modified GC solution Fl3can produce water-insoluble materials.

As you know, 5-ASA belongs to the class of nonsteroidal anti-inflammatory drugs (drug Mesalazine) and has the same properties as acetylsalicylic acid [Mashkovsky M. D. Medicines. - Kharkov: Torsing, 1997. - T. 2. - 590 c.]. As part of the modified GC 5-ASA may also exhibit anti-inflammatory properties. In connection with this the nutrient properties of the modified ha [Bondarev A. I. , Davydova C. A., Saudi F. C., Bashkatov S. A. anti-Inflammatory and analgesic activity of complexes of antiphlogistics with glycyrrhizic acid. // Pharmacology and toxicology. - 1991. - 5. - S. 47-50].

The study of anti-inflammatory properties were carried out on white rats weighing 180-200 g Experimental group rats were first treated with intraperitoneal administration of a solution of the modified SC at a dose of 60 mg/kg Over 30 min in the aponeurosis of the hind paws of rats were injected with 0.1 ml of 3% formalin, resulting developed swelling of the feet. A control group of rats remained untreated. After 3 h compared the foot with swelling with the foot without edema by measuring the amount of water displaced from the vessel when submerged feet to the bottom joint of the Shin bone. Statistically significant decrease swelling of the feet treated rats compared with control animals (P<0,05).

Experiments were performed on 21 mongrel dog weighing 10-15 kg Under hypentelium anesthesia was performed resection of the small intestine with the imposition of double-row anastomosis which are due to good adhesion of the film to the tissues of the colon. The animals were taken out of their experience on the 3rd, 7th and 30th day. The analysis of histological material, painted hematoxylineosin performed histochemical reaction to glycosaminoglycans Hale and glycogen (CHIC-reaction). Histochemical reactions were evaluated on a 4-point scale: inactive, weak, moderate and high activity.

Comparative results of morphological investigations are given in the table.

As follows from the above experimental results, the use of film explants based on hyaluronic acid promotes tissue regeneration, prevents inflammation and adhesions in the area of inter-intestinal anastomosis.

Modified hyaluronic acid can also be used for impregnation of the suture (surgical sutures, artificial vascular prostheses, etc.

The method of obtaining a modified hyaluronic acid, consisting of carbodiimide activation of the carboxylic groups of hyaluronic acid and subsequent reaction of the formed O-allodapini with a compound containing an amino group, characterized in that the hyaluronic acid first interacts with 1-(3-dimethylaminopropyl)-3-etilirovany acid in the initial reaction temperature 40oC for 2-4 h, after which the product cialiswhat against acidified distilled water, then purified on a column of aluminum oxide, precipitated with ethyl alcohol and dried under vacuum.


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