Polymeric compositions of modified hyaluronic acid and methods of using them

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

 

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the priority of Provisional Patent application U.S. No. 61/230074, registered July 30, 2009, and Provisional application for U.S. Patent No. 61/311953, registered on March 09, 2010, the content of which is given here by reference.

The technical FIELD TO WHICH the INVENTION RELATES.

The invention generally relates to a hyaluronic acid having a low level of modification of functional groups to the mixtures produced by the controlled reaction of this slightly modified hyaluronic acid with suitable bifunctionality or multifunctional reagents, and related to them the songs of the hydrogel and the hydrogel precursor. Described in the invention compositions are slightly crosslinked and have low Pro-inflammatory properties when introduced in vivo, and can be used along with other applications, such as medical devices, biomedical adhesives and sealants, and for delivery at a fixed place of bioactive substances.

The LEVEL of TECHNOLOGY

Hyaluronic acid is a natural anionic desulfation glycosaminoglycans, which is widely distributed in connective, epithelial and neural tissues. On average, the human body is ka with a body weight of 70 kg (154 pounds) contains approximately 15 grams of hyaluronic acid, the third of which is decomposed and synthesized) every day (Stern R., Euro J Cell Biol 83 (7): 317-25, (2004)). Since hyaluronic acid is found as a natural substance in many tissues of the body and, therefore, is biologically compatible, it is believed that it is well suited for applications in Biomedicine. And actually, many polymeric materials, including hyaluronic acid (also called hyaluronan), it derivateservlet form, and its conjugates can be used as injectable biomaterials, medical devices and implantable materials. Applications include delivery of molecules of therapeutic agent at a certain place, use as adhesives or sealants, tissue engineering, as viscoelastic supplements, and for wound healing. Hyaluronic acid, with the introduction and use as a therapeutic agent in its natural form, is usually quickly eliminated from the body, making necessary frequent injections. Typically, the polymer gel or the precursor gel may be appropriate properties, from the point of view of chemical reactions and conditions, gelation characteristics, and/or therapeutic effect in one or more in-vitro models, but in specific cases it is not possible to achieve a positive properties still is in vivo or therapeutic purposes.

The INVENTION

In the first aspect, features hyaluronic acid, modified on 10% or less in the reaction with diphenylsulfone. In particular, hyaluronic acid contains 10% or less of its hydroxyl groups, derivatizing by conducting the reactions proceed with diphenylsulfone.

In a specific embodiment, the hyaluronic acid is 1-10% of its hydroxyl groups, derivatizing 2-(vinylsulphonyl)ethoxy group. Get the activated hyaluronic acid with low levels of activation diphenylsulfone, in the description of the invention is usually called (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid or "g-GC".

In yet another embodiment, the hyaluronic acid has a degree of conversion of hydroxyl groups in 2-(vinylsulphonyl)ethoxy group selected from 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

In another more specific embodiment, the hyaluronic acid has a degree of conversion of hydroxyl groups in 2-(vinylsulphonyl)ethoxy group of about 4-5% on a recurring unit of the disaccharide.

In another embodiment, (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid has a molecular weight of from about 700 to about 3 million daltons.

In the second aspect, it is proposed hydrogel formed by re the functions (2-(vinylsulphonyl)ethoxy) 1-10%hyaluronic acid with tilenum cross-linking reagent having two or more tylnej groups.

In a related embodiment, tially cross-linking reagent has from two to about 8 tylnej groups. In yet another embodiment, tially cross-linking reagent has a number tylnej groups selected from 2, 3, 4, 5, 6, 7 and 8.

In yet another embodiment related to the second aspect, tilenum cross-linking reagent is functionalized by thiol polyethylene glycol (PEG) (PEG-thiol).

In an additional embodiment, the above, polietilenglikolya has a molecular weight from about 250 to about 20,000 daltons.

In a related embodiment, functionalized with thiol polyethylene is linear and has Tilney group at each end, that is, polietilenglikolya (PEG-dithiol).

In yet another embodiment, functionalized with thiol polyethylene glycol is a polymer with four chains in the form of diverging beams and has a core of pentaerythritol.

In yet another embodiment, functionalized with thiol polyethylene glycol polyol as one has a nucleus selected from glycerol dimer of glycerol (3,3'-oxydipropyl-1,2-diol)of trimethylolpropane, sorbitol, pentaerythritol, and g is cagliarini.

In an additional embodiment, the hydrogel formed by reaction (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid and tilenum cross-linking reagent, contains less than ten percent unreacted thiol and less than 10% unreacted vinylsulfonic groups. The number of remaining unreacted tylnej groups can be defined, for example, using the method and reagent of Ellman.

In yet another additional embodiment, the hydrogel formed by reaction (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid with tilenum cross-linking reagent, contains the mass percentage of polymer in water is from about 0.5 to 5.0 percent. In one or more implementation options, as an illustration of the contents of the polymer in water at a mass percentage of the obtained hydrogel choose 0,5, 1,0, 1,5, 2,0, 2,5, 3,0, 3,5, 4,0, 4,5 and 5 percent.

In yet another embodiment, the hydrogel formed by reaction (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid with tilenum cross-linking reagent is in the form of particles with a size of approximately from 0.10 to 3.0 millimeters.

In yet another embodiment, the above-mentioned hydrogel particles are in the form of a water suspension.

In yet another additional embodiment, the hydrogel described in either the bottom or more of the above embodiments, dispersed in an aqueous solution of unmodified hyaluronic acid.

In another more specific embodiment, a composition comprising cross-linked particles of the hydrogel in a solution of hyaluronic acid in physiological solution in which particles of the hydrogel formed by the reaction polietilenglikolya (PEG-dithiol) and hyaluronic acid having 1-10% of hydroxyl groups, derivatizing using 2-(vinylsulphonyl)ethoxy groups.

In yet another additional embodiment, the hydrogel described in any one or more of the above embodiments, includes a bioactive agent. In a particular embodiment, the bioactive agent is a corticosteroid. In another more specific embodiment, the bioactive agent is triamcinolone acetonide.

In yet another alternative embodiment, the hydrogel described in any one or more of the above embodiments, includes living cells.

In an additional embodiment, the hydrogel formed by reaction (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid with a bifunctional or multifunctional tilenum cross-linking reagent, shows low proinflammatory properties at NUTRISYSTEM the second injection in experimental models on the goats.

In a specific embodiment, a hydrogel exhibits a low Pro-inflammatory properties, as indicated by the response leukocyte reaction in the associated synovial fluid.

In another specific embodiment, a hydrogel exhibits a low Pro-inflammatory properties when intra-articular injection in experimental models on goats, as indicated by the calculation of the total points while conducting observations.

In yet another additional embodiment, the hydrogel is sterile.

In yet another additional embodiment, the invention hydrogel filled into the syringe.

In yet another additional embodiment, it is proposed a method of introducing any of the methods in the invention compositions of the hydrogel in the intra-articular space of a joint of a subject.

In addition, in the third aspect, features a method of obtaining (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid. The method involves the reaction of hyaluronic acid with diphenylsulfone under reaction conditions which ensure the reaction is not more than approximately 10% of hydroxyl groups on the repeating units of the disaccharide in hyaluronic acid with diphenylsulfone with the formation of (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid.

In a related embodiment, the implement is to be placed, the reaction involves the interaction of hyaluronic acid with a molar excess of diphenylsulfone.

In an additional embodiment, the step of the reaction is carried out in environmental conditions.

In yet another embodiment, the step of the reaction is conducted for from 10 seconds to approximately 120 seconds under the conditions of the environment.

In yet another embodiment, the step of the reaction is carried out in an aqueous solution of the base.

In an additional embodiment, the method further includes interrupting the reaction by adding acid. In a related embodiment, add appropriate amount of acid to adjust pH in the range from about 4 to 6.5.

In the fourth aspect described in the invention method is a method of producing a hydrogel. The method involves the reaction of (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid with tilenum cross-linking reagent having two or more tylnej groups under the reaction conditions which ensure the formation of cross-linked hydrogel. Suitable tirinya cross-linking reagents include functionalized by thiol polyethylene glycol, alkanethiol and other similar reagents.

In a related embodiment, the reaction is carried out at physiological pH value.

In another embodiment, re is the Ktsia performed in the absence of a polymerization initiator.

In yet another embodiment, the reaction is carried out without using an external source of energy.

In yet another embodiment, the reaction is carried out at a temperature in the range from 20°C to 45°C.

In yet another additional embodiment, the hydrogel comprises 10% or less of unreacted vinylsulfonic or tylnej groups. Preferably, the hydrogel consisted of 5% or less of unreacted sulfonic or tylnej groups. In a specific embodiment, the hydrogel practically does not include detectable unreacted vinylsulfonate or tirinya group.

In the fifth aspect, the kit comprising a syringe, where the syringe includes a hydrogel formed by reaction (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid with the above-described tilenum cross-linking reagent.

In yet another related embodiment, the syringe includes a hydrogel described in any one or more of the above embodiments, where the hydrogel is dispersed in an aqueous solution of unmodified hyaluronic acid. In a related embodiment, the aqueous solution is a saline solution.

In a related embodiment, the syringe has a form suitable for intra-articular injection of hydrogel using needle size 18-21.

p> In yet another related embodiment, the syringe includes a hydrogel described in any one or more of the above embodiments, where the hydrogel further includes a bioactive agent. In a related embodiment, the bioactive agent selected from the group consisting of steroids, growth factors, anti-proliferative funds, and antibiotics. In another more specific embodiment, the hydrogel comprises from about 0.01% to about 20% by weight of bioactive agent, of course, depending on the efficiency of the bioactive agent. In other words, the content of the less-active agent in the hydrogel is usually determined upper limit of the above interval, for example, approximately 10-20% by weight, while the content of potent bioactive agent will determine the lower bound of the interval, for example, from about 0.01 to 3% by weight. In a specific embodiment, in which the bioactive agent is triamcinolone acetonide, the hydrogel comprises from about 0.1 to 1% by weight of the bioactive agent.

In yet another embodiment, the syringe includes a hydrogel described in any one or more of the above embodiments, where the hydrogel further includes living cells. Examples of living cells is clucalc stem cells, parenchymal stem cells derived from blood cells and bone marrow cells.

In the sixth aspect, features a method of delivery of poorly water-soluble bioactive agent by introducing the subject described in the invention, a hydrogel comprising a poorly water-soluble bioactive agent dispersed in the hydrogel.

In the seventh aspect, describes a method for the treatment of acute and chronic inflammation associated with osteoarthritis, rheumatoid arthritis, other inflammatory arthritis, and with periodic application, by injection to a subject a hydrogel in accordance with any one or more of the described aspects of the invention, or variants of implementation in the intra-articular space of a joint such as the knee. In a particular embodiment, that is, when the hydrogel includes added corticosteroid, the method is effective from the point of view of the cartilage damage, which is reduced in comparison with cartilage damage occurring with the introduction of an equivalent amount of corticosteroid, is not included in the hydrogel, as shown at intra-articular injection in experimental models on the goats, using the total score on the assessment system Mancina through 28 days after injection. In a related embodiment, the above-mentioned method, that is, injection of hydro is the appropriate fields in the intra-articular space of a joint, is effective for pain relief in the subject compared to the pain experienced by the subject injections before him hydrogel. Usually, the subject begins to experience pain relief within about one hour to about one week after injection, more preferably, within about one hour to about 3 days after injection. In other words, pain relief usually occurs within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after injection, or, if not within the first twenty-four hours, during 1, 2, 3, 4, 5, 6, or 7 days after injection. Usually, it is assumed that the period of pain relief ranged from approximately 3 to 9 months, that is, from 3, 4, 5, 6, 7, 8, 9 months or even more.

In the eighth aspect, features a method of reducing damage to the cartilage in the introduction suffering from osteoarthritis subject of corticosteroid in the intra-articular space of a joint by adding the corticosteroid crosslinked hydrogel prior to the introduction or introduction to the subject. Crosslinked hydrogel is typically a hydrogel based on hyaluronic acid, which will be described in more detail below. An example of a cross-linked hydrogel is a hydrogel, which is hyaluronic acid, modified to the extent of 10% or less by reaction with divas is Nursultanom and then stitched using Colnago cross-linking reagent, having two or more tylnej groups. Surprisingly the inclusion of corticosteroid crosslinked hydrogel is less cartilage damage than in the case of the introduction of an equivalent dose of corticosteroid, without including it in the hydrogel.

In the ninth aspect, related to the above aspects, the method of treating osteoarthritis by introducing to the subject a therapeutically effective amount of corticosteroid in the intra-articular space of a joint, the invention offers improvements, including the introduction of corticosteroid in the form of compositions crosslinked hydrogel, comprising a corticosteroid, which in turn decreases the degree of damage to cartilage than in the case of the introduction of an equivalent dose of corticosteroid, without including it in the hydrogel.

In a specific embodiment relating to the seventh, eighth and ninth aspects, a corticosteroid selected from the group consisting of hydrocortisone, hydrocortisone acetate, cortisone acetate, pivalate tixocortol, prednisolone, methylprednisolone, prednisone, triamcinolone, salt, triamcinolone, such as triamcinolone acetonide, triamcinolone benetone, triamcinolone puretone, triamcinolone hexacetonide, triamcinolone diacetate, triamcinolone alcohol, mometasone, amcinonide, budezonida, desonide, fluocinonide, fluorine what she acetonide, halcinonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, alclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone kaproata, fluocortolone pivalate, fluprednidene acetate, monohydrate beclomethasone dipropionate, flunisolide, fluticasone propionate, monohydrate mometasone furoate and fluticasone furoate.

In another more specific embodiment, the corticosteroid is triamcinolone acetonide.

In the tenth aspect, it is proposed dosage form comprising a poorly water soluble drug such as a steroid, enclosed in a three-dimensional structure described in the invention of the hydrogel, and the injection of such dosage forms in the intra-articular space of a joint.

In one embodiment, related to the above aspects, the inclusion of particles of steroid inside the hydrogel is an effective measure to prevent direct contact of the majority of the particles of the steroid with the tissues of the joint.

In yet another related embodiment, the inclusion of particles of steroid inside the hydrogel is an effective measure to maximize the locale is used the concentration of steroid in the joint and minimize its total concentration in the body.

In yet another additional embodiment, the inclusion of particles of steroid inside the hydrogel is an effective measure to protect the particles of the steroid from the premature removal from the joint.

In yet another additional embodiment, inclusion of the steroid in the hydrogel, therapeutic efficacy of steroid is achieved at a lower total dose than in the case when not in use turn steroid in the hydrogel, and thus minimizes unwanted local and systemic side effects.

In a related aspect, it is proposed the use described in the invention hydrogel for injection or implantation near or in bone, teeth, nerves, cartilage, blood vessels, soft tissue, or other tissue of a mammal.

Additional embodiments of the compositions, methods, kits, and other implementations will become apparent from the following description, examples and claims. From the above and subsequent description it is obvious that all, without exception, described in the invention of distinctive features, and all, without exception, a combination of two or more of these distinctive characteristics, are included in the scope of the present invention, provided that the distinctive features included in such combination are not mutually inconsistent. In addition, Liu is Oh distinctive characteristic or combination of distinctive features can be specifically excluded from any variant of implementation of the present invention. Additional aspects and advantages of the present invention are outlined in the following description, particularly when considered in conjunction with the accompanying description, examples and drawings.

BRIEF DESCRIPTION of DRAWINGS

In FIG. 1 shows the range of1H NMR modified vinylsulfonate hyaluronic acid (ha-sun), obtained as described in Example 1. Using NMR, it was determined that hyaluronic acid has a degree vinylsulfonate replacing approximately four percent of the disaccharide.

In FIG. 2 shows the graphical dependence of the percentage release of poorly water-soluble model drug, triamcinolone acetonide, the serial number of samples, the preparation of which is described in Example 16.

In FIG. 3 shows a graphical dependence released the total mass of poorly water-soluble model drug, triamcinolone acetonide, the serial number of samples, the preparation of which is described in Example 16.

In FIG. 4 shows a graphical dependence of the number of redundant triamcinolone acetonide from the sequence numbers of samples, the preparation of which is described in Example 16.

In FIG. 5 graphically presents the number of leukocytes in the synovial fluid (the number of cells in a cubic millimeter) in the tribe of goats, to the e were injected with the test material 1, with regard to the group subjected to treatment with other examinees materials, assessed 24 hours after injection of 1.5 ml described in Example 17. The test material 1=GK-VS/PEG-(SH)2the gel.

In FIG. 6 graphically presents the absolute number of leukocytes in synovial fluid (absolute amount=total amount × the number of leukocytes in the synovial fluid in the knee goats, which were injected with the test material 1, in relation to the group subjected to treatment with other examinees materials, assessed 24 hours after injection of 1.5 ml described in Example 17. The test material 1=GK-VS/PEG-(SH)2the gel.

In FIG. 7 graphically presents the differential distribution of cells from the synovial fluid (average for group) in the knee is subjected to injection goats in relation to the group subjected to treatment with other examinees materials, assessed 24 hours after injection of 1.5 ml described in Example 17. For each test material shows the distribution polymorphically leukocytes (PMN), lymphocytes, monocytes and eosinophils (Eos).

In FIG. 8 graphically presents the average total scores for synovial fluid, joint tissue, and the joint points of the synovial fluid and joint tissues (table 6) for the knees subjected to injection goats for each tipin the th of the test material, described in Example 17, where the Total number of points=Number of points for synovial fluid+the total number of points for the joint. The maximum number of points for synovial fluid or the total number of points for the joint 8 is at the value 0 in the norm; the maximum number of points for the total number of points is 16 when the value 0 in norm.

In FIG. 9 shows the scoring of the degree of staining dye safranin O for samples of cartilage of the joints goats subjected to treatment with the tested materials are described in detail in Example 34, 14 days after injection. The test material 1: GK-VS-PEG-(SH)2Person 2 material: GK-VS-PEG-(SH)2-TA.

In FIG. 10 shows the scoring of the degree of staining dye safranin O for samples of cartilage of the joints goats subjected to treatment with the tested materials are described in detail in Example 34, at 28 days after injection.

In FIG. 11 shows the calculation results of the number of points on the evaluation Mancina for samples of cartilage of the joints goats subjected to treatment with the tested materials are described in detail in Example 34, at 28 days after injection.

In FIG. 12 and FIG. 13 shows the characteristic histological preparations of the medial femoral condyle, painted Safra is another O (40X), after 14 days (Fig. 12) and 28 days (Fig. 13) after injection, respectively, described in detail in Example 34.

In FIG. 14 graphically presents the average number of leukocytes in synovial fluid (mean+standard deviation) for all of the animal relative to the test material and Control material, assessed 24 hours after intra-articular injection volume of 1.5 ml, as described in detail in Example 45.

DETAILED description of the INVENTION

Further, the present invention is described in more detail. However, this invention can be implemented in many different forms, and are listed in the description of the invention embodiments of should not be construed as limitations to the invention; moreover, these implementation options are presented in order to fully and completely disclose to specialists in this area, the nature and scope of the invention.

The contents of all cited above or below in the description of the publications, patents and patent applications, is contained in the description by reference, unless otherwise noted. When the same term is defined in the publication, patent or patent application, the content of which is by reference to them, and in the description of the present invention, preference should be given to the definition given is the description of the present invention. For publications, patents, and patent applications mentioned in connection with the description of a particular type of connection, chemistry, and so on, parts of the document relating to such compounds, the chemistry, and so on, are such parts, the content of which is contained in the description by reference.

Definitions

It should be noted that in this description, the singular number include the designation of multiple objects, if it is not explicitly contradicts the meaning of the content. So, for example, reference to "a polymer" includes a single polymer, but also two or more identical or different from each other polymers.

Unless specifically stated otherwise, the definitions in the invention are standard definitions used in the field of organic synthesis, polymers and pharmaceuticals.

In the description and in the claims of the present invention will use the following terminology in accordance with the below definitions.

"Biocompatible polymer" is a polymer whose degradation products are compatible with living tissue, or which may have useful biological properties. The biocompatible polymer may be biocompatible itself, and/or may be synergistic biocompatible when used and in combination with a biologically active agent.

The term "polymer hyaluronic acid" refers to a polymer that includes a repeating disaccharide glycosides of poznania hyaluronan, where the repeating unit can be derivationally one or more of the provisions of D-glucuronic acid and/or D-N-acetylglucosaminidase link repeating disaccharide glycosides of subzone. It is assumed that the term "polymer hyaluronic acid" includes hyaluronic acid (also called hyaluronan), derivational hyaluronic acid, salt forms, complexes of hyaluronic acid with cross-linking reagents and conjugates of hyaluronic acid. It is assumed that the term "hyaluronic acid" refers to unmodified or noderivatives hyaluronic acid.

The terms "derivative of hyaluronic acid" or "derivational hyaluronic acid" or "modified hyaluronic acid" refers to hyaluronic acid, which was derivatization in the reaction, for example, with one or more small chemical units, such as diphenylsulfone or other similar links.

Derivationally the thiol polymer hyaluronic acid refers to the above-described polymer of hyaluronic acid having three or more repeating units of the disaccharide and comprising at least one sulfhydryl (Tilney) GRU is PU.

The term "reactive" refers to a functional group (for example, present in the polymer), which under normal conditions of organic synthesis reacts easily or is involved in the reaction at a reasonable rate. This group is opposed to those groups who either do not respond or require the use of active catalysts or create almost unreal the reaction conditions for the interactions (i.e., "directionspanel" or "inert" group).

Used in the description of the invention, the term "molecular weight or molecular weight with respect to the water-soluble polymer, such as hyaluronic acid, refers to a conditional srednekamennogo molecular weight of the polymer, determined using the method of multiple-angle light scattering. Molecular weight can be expressed either as srednekislye molecular weight, or as srednevekovaja molecular weight. Unless otherwise specified, all references in the invention, molecular weight refers to srednekamennogo molecular weight.

The term "hydrogel" refers to water containing a three-dimensional hydrophilic polymer structure or gel in which water is the dispersion phase and in which the water content is more than 50 wt.%. Described in the invention Hydra the gels usually do not require initiators or accelerators stitching to achieve the desired degree of crosslinking.

"Sterile" composition is a composition that does not contain viable microbes, which is determined using samples for sterility in accordance with the requirements of the U.S. Pharmacopoeia. See the document "The United States Pharmacopeia", 30th Revision, The United States Pharmacopeial Convention: 2008.

Used in the description of the invention, the term "lightly crosslinked" or "having a low degree of crosslinking" means that when the reaction of crosslinking with the formation of the final crosslinked gels in the reaction are from about 40% to about 100% available for stitching seats, while the modified hyaluronic acid, used as starting material for the formation of a gel, has 10% or less of its hydroxyl groups in the activated/derivateservlet form the hydrogel, which is generally regarded as slightly crosslinked.

Hydrogel that exhibits low Pro-inflammatory properties when intra-articular injection in experimental models on goats, is a hydrogel that in the study described in the invention of intra-articular injection in experimental models goats characterized by the number of cells in the synovial fluid of less than 20,000 cells per cubic millimeter after 24 hours after injection, and it is preferable that the number of leukocytes in the synovial liquids and amounted to less than 15,000 cells per cubic millimeter after 24 hours after injection, where the number of leukocytes is an average of three samples taken from three individually injected animals.

Containing corticosteroid hydrogel, which reduces cartilage damage or which results in less cartilage damage than an equivalent dose of corticosteroid, enter the subject in the absence of hydrogel, usually characterized using any suitable model to assess the damage to the cartilage, but it is preferable to carry out its assessment are described in detail in the invention injections in the knee in an experimental in vivo model for the goats. The experimental data is generally collected at least 7 days after injection, but not more than 28 days after injection. The preferred system of assessment is the total number of points on the evaluation Mancina; material, which reduces cartilage damage, estimated as described above, compared with the use of only one medication is a material which is characterized by improved average scores compared to the drug (i.e., a corticosteroid) that is administered in an equivalent amount. Preferably, the total number of points on the evaluation Mancina for included in the hydrogel of medicines has improved, at least at one or more points on compared the Yu and the total number of points on the evaluation Mancina for medicines entered without its inclusion in the hydrogel.

Used equivalent terms "drug" or "pharmaceutically active agent" or "bioactive agent" or "active agent" refers to any organic or inorganic compound or substance having bioactivity and adapted or used for therapeutic purposes. The broader notion of "medicinal product" includes proteins, hormones, anti-cancer tools, synthesized chemical compounds and mimetics, oligonucleotides, DNA, RNA and products of genetic engineering. It is assumed that the reference used in the description of the invention the drug, as well as reference to other chemical compounds, as appropriate, includes a connection to any one of its pharmaceutically acceptable forms, including isomers such as diastereomers and enantiomers, salts, solvate and polymorph, specific crystalline forms as well as racemic mixtures and pure isomers described in the invention compounds.

Used in the description of the invention, the term "solid" refers to non-leaking substance, including crystalline forms, polymorphs, noncrystalline amorphous substances, precipitation, particle, or other similar solids. Each of these solid forms can have different sizes, approximately the nutrient from 0.01 microns to 2000 microns, for example, from about 0.01 microns to 1 microns, from 1 micron to 100 microns; from 100 microns to 1000 microns, 1000 microns to 2,000 microns, from about 1,100 microns to 1500 microns and 1500 microns to 2000 microns.

Here is the description of the invention, the particle sizes are diameters of the particles and are usually determined using sieve analysis. The described dimensions or ranges generally correspond to the size of the holes in the sieve or grid. You can use a table to translate the size of the particles from one system to another, for example, in millimeters from the corresponding specific values of the mesh or sieve. See, for example, Examples 39 and 40.

"Water-insoluble drug" or "poorly water-soluble drug is a drug having a solubility in water below 10 mg/mg.

Given in the description of the invention, the terms "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" of a composition or hydrogel or polymer), refer to a nontoxic but sufficient amount of the composition to provide the desired response, such as prevention, reduction, or elimination of pain in the subject. The exact quantity required will vary from subject to subject, depending on the species, age and General state of health of the subject is and, the severity of the condition being treated, used a given drug or funds, the specific characteristics of the composition, the route of administration and other factors. In any given case, an appropriate "effective" amount can be determined by conventional expert in this area by carrying out the appropriate experiment.

"Treating" or "treatment of" acute or subacute pain include: pain suppression, that is, the suspension of normal development or return of pain, or pain relief, that is, reducing the force of pain experienced by the subject.

"Optional" or "optionally" means that the hereinafter described circumstance may occur or may not occur, so that the description includes instances in which circumstance occurs and instances when it does not occur.

The term "significantly" in relation to a particular characteristic or value involves a significant degree or almost full value (i.e., the degree of 85% or more) in relation to a particular characteristic or value.

It is assumed that the term "approximately", especially in relation to this number includes the deviation of plus or minus five percent.

In the following sections can also meet additional definitions.

A brief overview

This application is based at least in part, on the discovery by the inventors of the hydrogels with extremely low proinflammatory properties when administered in vivo. When conducting research related to the present invention, the inventors have found that many of biocompatible hydrogels with seemingly favorable chemical, rheological and other physical properties, and which manifest themselves positively in a number of biocompatible and conventional in-vitro and in-vivo models, can cause inflammation and pain, particularly when intra-articular injection. In the study of injection in the joint on the experimental model on goats and comparison with similar Hydrogenium compositions, it was found that described in the invention, the materials have extremely low proinflammatory properties. See, e.g., Example 17 and Figure 4-8. Usually, the hydrogels of the present invention when introduced into the intra-articular space of a joint (for example, in the study of injection in the joint in an experimental model of the goats) were characterized by reduced negative or unwanted side effects on cartilage, compared with the introduction in the intra-articular space of a joint or the equivalent quantity produced by the industry viscoelastic supplements, or introduction is receiving an equivalent amount of active reagent, not included in the described in the invention, the hydrogel.

It has been unexpectedly found that the inclusion of corticosteroid crosslinked hydrogel, such as described in the invention, in fact leads to less damage of the cartilage than in the case of the introduction of equivalent or higher doses of corticosteroid, is not contained in the hydrogel. For an example, see Example 34 and Figure 9-13. In addition, the invention results, indicating that intra-articular injection of the hydrogels of the present invention does not provide local or systemic effects nor with the introduction of them by themselves (that is, in the absence of the active agent), nor when introduced in combination with a corticosteroid, such as triamcinolone acetonide.

Described in the invention of the exceptional properties of the hydrogels usually get by controlled reaction of hyaluronic acid with clearly defined low degree of modification of the functional groups with suitable bifunctionality or multifunctional cross-linking reagents. The resulting hydrogels get under mild conditions without the use of initiators or accelerators or other harmful additives. It is assumed that the resulting hydrogels will have the minimum number of unreacted reactive groups, and get them from the minimum number of reagents and the reaction compound is. Hydrogels crosslinked, and it was also shown that they can be used to capture and release of bioactive agents in slow and steady in time mode. For an example, see Figure 2-3.

The characteristic features of the compositions, methods and kits, and other similar questions are discussed in more detail below.

Polymers derivateservlet hyaluronic acid

The hydrogels of the present invention can be formed from various polymeric materials. Preferred are biodegradable or biopolymere polymers, modified to a certain extent the content of one or more reactive functionalities. Preferably, the polymer was polyanionic polysaccharide (PAS). Non-limiting examples of polyanionic polysaccharides include, for example, hyaluronic acid (ha), carboxymethylcellulose (CMC), carboxymethylamino (KMA), chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, dermatan 6-sulfate, heparin sulfate, heparin, keratin sulfate and their derivatives, and combinations thereof. Such polymers are known in the art and described, for example, in U.S. Patent No. 6056970. Other biodegradable polymers include fibrin, fibrinogen, starch, polyaminoamide; peptides, proteins, gelatin and collagen.

The preferred polymer is hyaluronic acid, which is called the I-hyaluronan. Hyaluronic acid is a natural linear polysaccharide consisting of alternating disaccharide glycosides parts of N-acetyl-D-glucosamine and D-glucuronic acid, the United alternating β 1->3 glucuronid and β 1->4 glucosaminidase relationships, resulting in duplicate link is (1->4)-β-D-GlcA-(1->3)-β-D-GlcNAc. Hyaluronic acid for use in the preparation of one or more of the tested hydrogels usually derivatized with one or more reactive groups, such as vinylsulfonate, acrylate, methacrylate, and other similar groups. Preferably, the hyaluronic acid was derivateservlet with only one reactive group. The degree of modification or derivatization may be in the range from 1% to 100% modification of the reactive functional groups in the polymer, although a low degree of modification of the polymer are usually preferred.

One example of a modified hyaluronic acid is hyaluronic acid, derivatization in the reaction of its hydroxyl groups with diphenylsulfone. Hyaluronic acid may typically have a degree of modification of the reactive hydroxyl groups in the range from about 1 to about 80%. In other words, a 1% degree of modification or substitution of oznachaet is, on average, 1% of disaccharide glycosides links hyaluronic acid contains vinylsulfonate group. Preferably, hyaluronic acid would have a degree of modification of the reactive hydroxyl groups approximately in the range of 1-50%. More preferably, hyaluronic acid would have a degree of modification of the reactive hydroxyl groups in the range from about 1 to about 25%. In a specific embodiment, hyaluronic acid modify to the extent of 10% or less by reaction with diphenylsulfone. In particular, in the preferred embodiment, the hyaluronic acid is 10% or less of hydroxyl groups, derivatizing in the reaction of accession with diphenylsulfone. Hydroxyl groups of hyaluronic acid are transformed into (2-(vinylsulphonyl)ethoxy) group. The obtained activated hyaluronic acid is usually called in the description of the invention (2-(vinylsulphonyl)ethoxy)hyaluronic acid or g-GC. In particular, hyaluronic acid may have a degree of conversion of hydroxyl groups in (2-(vinylsulphonyl)ethoxy) groups selected from the following values: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%. Alternatively, hyaluronic acid may have a degree of conversion of hydroxyl groups, which is the interval between any two given to enter the percent: for example, 1-10%, 2-10%, 3-10%, 4-10% and so on for all combinations of the given integers, for example, 2-7%, 2-6%, 3-8%, 3-7% and so on. In another more specific embodiment, the hyaluronic acid has a degree of conversion of hydroxyl groups in (2-(vinylsulphonyl)ethoxy) groups of approximately 4-5% of the repeating disaccharide glycosides link. In specific cases, the level of modification of the functional groups of the hyaluronic acid is clearly stated (i.e., defined) in order to adjust and optimize the concentration of cross-linking reagent, along with other parameters that allows to regulate the subsequent crosslinking reaction. The degree of substitution/modification of the original polymer can be determined using any of a number of appropriate techniques, such as NMR, UV or IR analysis, or elemental analysis. The preferred method of calculating the percentage of substitution of the polymer, such as hyaluronic acid, is NMR, for example, proton NMR. See, for example, Example 1, in which the degree of modification of the hyaluronic acid was determined based on the ratio of the relative peak areas corresponding to vinylsulfonate and acetamidomethyl group of hyaluronic acid in the spectrum of1H NMR.

The polymer may also include hydrazide reactive group and/or aminooxy reactionactos the groups, described in the patent document PCT/US/2004/040726 (WO 2005/056608), while the content of the relevant parts of this invention associated with the derivatization of such polymers themselves in the obtained polymers are given here by reference.

Alternatively, the polymer may be a thiol-derivational, such as thiol-derivativesa hyaluronic acid. Examples of polymers with thiol-derivateservlet hyaluronic acid include the examples described in U.S. Patents№№ 6884788; 6620927; 6548081; 6537979; 6013679; 5502081 and 5356883, while the content of the relevant portions of which are connected with such thiol-derivationally polymers are given here by reference.

Additional examples of polymers of hyaluronic acid include cysteine-derivational hyaluronic acid, including but not limited to, those polymers, which are described in the publication "Controlled Release from Glycosaminoglycan Drug Complexes" R. V. Sparer et al., Chapter 6, pages 107-119, in T. J. Roseman et al., CONTROLLED RELEASE DELIVERY SYSTEMS, Marcel Dekker, Inc., New York (1983).

Examples of additional preferred polymers include hyaluronic acid, derivatisation using suspended Tilney group associated with N-allocating group through hydrocarbonous, aryl, substituted hydrocarbonous, or substituted aryl group. Illustration of polymers for use in the proposed invented the eat compositions and methods include Carbylan™-S (described in detail in patent document International Patent Publication No. WO 2005/056608).

Additional derivateservlet polymers include hyaluronic acid, covalently linked to a reactive cross-linking reagent, such as difunctional or multifunctional acrylate, allyl or methacrylate compound. Typical cross-linking reagents for modification of hyaluronic acid include, but are not limited to, polyethylene-glycol diacrylate (PEGDE), polyethylene glycol dimethacrylate (PGDM), polyethylene-glycol diacrylate (PEGDE) and polyethylene-glycol dimethacrylate (EGDMA) and their derivatives. PEG-links above cross-linking reagents can be oligomeric or polymeric, for example, comprising from 2 to 100 or more pozvani. Additional cross-linking reagents suitable for the modification/functionalization of the polymer, such as hyaluronic acid, include dextran acrylate, methacrylate dextran, glycidyl methacrylate dextran, functionalized methacrylate hyaluronic acid functionalized acrylate hyaluronic acid, glycerin dimethacrylate, 1,3-diglyceride diacrylate glycerin, sorbitol acrylate and derivatives thereof.

Derivativesa hyaluronic acid or other polymer can typically have srednekamennogo molecular weight in the range from about 700 to 3000000 daltons. Illustrations intervals Molek is Arnau masses are the intervals from about 1000 up to 2,000,000 daltons, or from about 5000 to 1,000,000 daltons. Additional suitable intervals molecular masses include intervals from about 50,000 daltons to about 1000000 daltons, or from about 100,000 daltons to about 1200000 daltons, or from about 90000 daltons to approximately 300,000 daltons. The molecular weight of hyaluronic acid are usually the value srednekamennogo molecular weight, which can be determined, for example, by using exclusive chromatography detector the multiple-angle laser light scattering (MALLS-SEC). Depending on the origin hyaluronic acid may have a polydispersity (Mw/Mn) to about 3, or more preferably, up to about 2. Usually, hyaluronic acid as starting material can have a fairly narrow molecular weight distribution, with values less than about 2.5, more preferably less than about 2. Examples of intervals polydispersity hyaluronic acid include interval approximately from 1,02 to about 2.5, where the source of hyaluronic acid may have a polydispersity of approximately 1,02, 1,05, 1,1, 1,2, 1,3, 1,3, 1,5, 1,6, 1,7, 1,8, 1,9, 2,0, 2,1, 2,2, 2,3, 2,4 or 2.5, or even higher. Alternatively, suitable as a starting material d is I derivatization hyaluronic acid may have a viscosity, usually centipoise, with specific concentration in water, which corresponds to any one or more of the above intervals srednekamennogo molecular weight.

Crosslinking reagent

Examples of cross-linking reagents that are effective in the formation of hydrogels having described the invention preferred indications include compounds having two or more reactive groups located on the Central molecule "C". The Central molecule may be linear or cyclic alkane, oligomeric or polymeric PEG, or any other such suitable Central molecule. In the case of cross-linking reagents on the basis of the PEG, the peg may be linear, branched (polymer having two branches), or mnogochislennym (for example, having 3, 4, 5, 6, 7, 8 or more polymeric branching). Therefore, in such cases, the Central molecule can usually be linear PEG, branched PEG having 2 divisions or mnogorazemny PEG having branches PEG emanating from the Central core. Illustration cores for such mnohorozmernych polymers include aritra, pentaerythritol, trimethylolpropane, glycerol dimer of glycerol (3,3'-oxydipropyl-1,2-diol), oligomers of glycerol, sorbitol, hexaglycine, and other such compounds.

For example, cross-linking R the agent may be a Central molecule "C", having located therein tirinya or acrylate groups. Tilligerry cross-linking reagent comprises two or more tylnej group. Such tirinya groups can react with vinylsulfonate, for example, inside derivateservlet vinylsulfonate hyaluronic acid. Illustration tylnej cross-linking reagents include PEG-dithiol (HS-PEG-SH, PEG-trithiol with 3 branches (glycerol core), PEG-titration with 4 branches (pentaerythritol core), or PEG-occation with 8 branches (hexaglycine core). The above reagents from mnogochislennogo PEG may also be not all branching functionalityand using thiol. Additional suitable tirinya reagents with PEG as a Central molecule, produced by the firm Laysan Bio (Arab, Alabama), as well as aromatic dithioles produced by the firm NanoScience. Other suitable tirinya cross-linking reagents include dimercaptan acid, 2,3-dimercapto-1-propanesulfonic acid, dihydrolipoic acid, functionalized with thiol acid and functionalized by thiol hyaluronic acid. Can also be used cross-linking reagents having terminal acrylate group, located on the Central molecule. For example, suitable for use as cross-linking reagents are described above cent is real molecules, in which tirinya group substituted on acylate or methacrylate group. Additional examples of cross-linking reagents include the examples described in the patent document PCT/US/2004/040726.

Cross-linking reagents also include molecules containing acrylate, allyl or methacrylate group. Acrylate, allyl or methacrylate cross-linking reagents may be in the nature of a Monomeric or polymeric. In one embodiment, cross-linking reagent selected from the group including polyethylene-glycol diacrylate (PEGDE), polyethylene glycol dimethacrylate (PGDM), polyethylene-glycol diacrylate (PEGDE) and polyethylene-glycol dimethacrylate (EGDMA), dextran acrylate, methacrylate dextran, glycidyl methacrylate dextran, functionalized methacrylate hyaluronic acid functionalized acrylate hyaluronic acid, glycerin dimethacrylate, 1,3-diglyceride diacrylate glycerin, sorbitol acrylate and derivatives thereof.

Molecular weight cross-linking reagent is usually smaller than the molecular weight of the above modified hyaluronic acid or other polymer. Typically, the molecular weight of the crosslinking reagent is in the range from about 200 to about 20,000 daltons. Additional examples of intervals molecular masses for cross-linking reagent are online raly from about 1000 to about 10000 daltons (e.g., having a molecular weight of 1 KD, 2 KD, 3 KD, 4 KD, 5 KD, 6 KD, 7 KD, 8 KD, 9 KD or 10 KD, where KD denotes kilodaltons) or even from about 1000 to 5000 daltons. Examples of molecular weights for cross-linking reagent, such as the PEG dithiol, or any of the other above-described suitable cross-linking reagents include, among other approximately 3350, 3400 and 5000 daltons.

Bioactive agents

The invention hydrogels, predecessors hydrogels, and related compositions and/or kits can optionally include a bioactive agent. Bioactive agents that can be entered in the invention compositions and combinations that include antimicrobial agents, antibiotics, analgesics, anti-proliferative/antimitoticescoy tools, including natural products such as Vinca alkaloids (e.g. vinblastine, vincristine, and vinorelbine), paclitaxel, epileptogenesis (e.g., etoposide, teniposide), antibiotics (dactinomycin (actinomycin D) daunorubicin, doxorubicin and idarubitsin), anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin, enzymes (L-asparaginase); antiproliferative/antimitoticescoy alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamine (g is camerinelli, thiotepa), the alkyl sulphonates - busulfan, nitrosoanatabine (carmustin (BCNU) and analogs, streptozocin), trazeni - dacarbazine (DTIC); antiproliferative/antimitoticescoe antimetabolites, such as analogs of folic acid (methotrexate), pyrimidine analogues (fluorouracil, floxuridine, and cytarabine), purine analogues and related inhibitors (mercaptopurine, tioguanin, pentostatin and 2-chloromethoxypropyl [cladribine]); coordination complexes of platinum (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutetimid; hormones (e.g. estrogen); anticoagulants (heparin, salt synthetic heparin and other inhibitors of thrombin); fibrinolytic tools (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-migration of tumor cells; antisecretory agents (such as brefeldin A); anti-inflammatory agents, such as adrenal cortical steroids (hydrocortisone, hydrocortisone acetate, cortisone acetate, pivalate tixocortol, prednisolone, methylprednisolone, prednisone, triamcinolone acetonide (or any other pharmaceutically acceptable salt of triamcinolone), triamcinolone alcohol, mometazon, amcinonide, budesonide, desonide, fluocinonide, fluotsinolon acetonide, halcinonide, betamethasone, betamethasone NAT is the third phosphate, dexamethasone, dexamethasone sodium phosphate, and fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, alclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and flupredniden acetate. The monohydrate of beclomethasone dipropionate, flunisolide, fluticasone propionate monohydrate mometasone furoate, triamcinolone acetonide, fluticasone furoate, non-steroidal drugs (salicylic acid derivatives, for example, aspirin); derivatives of paraaminophenol, i.e., acetaminophen; indole and indeloxazine acid (indomethacin, sulindac and etodolac), heteroarylboronic acid (tolmetin, diclofenac, and Ketorolac), arylpropionic acid (ibuprofen and derivatives), Anthranilic acid (mefenamico acid and meclofenamic acid), enol acids (piroxicam, tenoxicam, phenylbutazone and occidentalise), nabumeton, gold compounds (auranofin, aurothioglucose, sodium thiomalate gold); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mofetil mycophenolate); mitogenic or morphogenetic proteins, peptides or mimetics growth factor; growth factor vascular endothelial (VEGF), fibroblast growth factor (FGF), members of the superfamily of transforming growth factor is-β (TGF-β), including TGF-β and bone morphogenetic proteins (BMP) such as BMP-2, 3, 4, 5, 6, 7, 8; growth factor, insulin and insulin-like growth factor (IGF), a growth factor for hepatocytes (HGF), growth factors, epidermal (EGF), protein family Hedgehog (SHH and IHH), activin, inhibin, demineralized bone matrix (DBM) and growth factors, platelets (PDGF's), hematopoietic growth factors (G-CSF, CSF-1, GM-CSF, erythropoietin, cytokines and lymphokines, including the family of interleukins (IL-1 to IL-34)), interferons, nerve growth factors (NGF), neutralizing, antagonistic or agonistic antibodies, agonists or antagonists of growth factor receptor, nitric oxide donors; antisense oligonucleotides, transcription factors, mediators of signaling cascades, and combinations thereof.

Antibiotics include antibiotics of the lincomycin family (belonging to the class of antibiotics, initially extracted from the bacteria Streptomyces Lincolnensis); antibiotics of the tetracycline family (belonging to the class of antibiotics, initially extracted from the bacteria Streptomyces Aureofaciens); antibiotics on the basis of sulfur-containing compounds such as sulfonamides; and other similar. Examples of antibiotics of the lincomycin family include the lincomycin (6,8-dideoxy-6-[[(1-methyl-4-propyl-2-pyrrolidinyl)carbonyl]amino]-1-thio-L-threo-α-D-galactocerebroside), clindamycin, 7-deoxy, 7-chloro derivative of lincomycin (n is an example, 7-chloro-6,7,8-trideoxy-6-[[(1-methyl-4-propyl-2-pyrrolidinyl)carbonyl]amino]-1-thio-L-threo-α-D-galactocerebroside), and their pharmacologically acceptable salts and esters. Examples of antibiotics of the tetracycline family include the tetracycline 4-(dimethylamino)-1,4,4 α,5,5 α,6,11,12 α-octahydro-3,6,12,12 α-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide), chlortetracycline, oxytetracycline, tetracycline, demeclocycline, rolitetracycline, metatsiklina and doxycycline and their pharmaceutically acceptable salts and esters, in particular, salts of accession acids, such as cleaners containing hydrochloride salt. Examples of antibiotics on the basis of sulfur-containing compounds include, but are not limited to, sulfonamides, sulfacetamide, sulfabenzamide, sulfadiazine, sulfadoxine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole, and their pharmacologically acceptable salts and esters, for example, sulfacetamide sodium. Antimicrobial agents and/or antibiotics may additionally include compounds such as erythromycin, bacitracin, neomycin, penicillin, polymyxin B, tetracyclines, viomycin, Chloromycetin and streptomycin, Cefazolin, ampicillin, azactam, tobramycin, clindamycin and gentamicin.

Analgesics include compounds such as lidocaine, benzocaine and marcain.

By the invention, the hydrogel may also include living cells. Examples of living Clearblue stem cells, parenchymal stem cells isolated from the blood cells and bone marrow cells.

In one preferred embodiment, the hydrogel comprises a corticosteroid. Examples of suitable corticosteroids include hydrocortisone, hydrocortisone acetate, cortisone acetate, pivalate tixocortol, prednisolone, methylprednisolone, prednisone, triamcinolone, salt, triamcinolone, such as triamcinolone acetonide, benetone triamcinolone, puretone triamcinolone, hexacetonide triamcinolone, triamcinolone diacetate, triamcinolone alcohol, mometazon, amcinonide, budesonide, desonide, fluocinonide, acetonide fluoqinolona, halcinonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, dipropionate of alclometasone, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, caproate of fluocortolone, pialat of fluocortolone, acetate of fluprednidene, monoidal of beclomethasone dipropionate, flunisolide, fluticasone propionate monohydrate mometasone furoate and fluticasone furoate.

One preferred compound for use in the dosage form in the form of the invention the hydrogel is triamcinolone (11β,16α)-9-fluoro-11,16,17,21-tetrahydroxypregna-1,4-diene-320-dione), or its pharmaceutically acceptable salt or MES. Structure triamcinolone acetonide shown below.

The bioactive agent may usually be mixed with the hydrogel, suspended in the hydrogel, or enclosed inside the invention of the hydrogel. Alternatively, the bioactive agent can be in the form of a polymer conjugate, or may be covalently bonded, but with the further possibility of release, with the component used to prepare the hydrogel, for example, with a modified hyaluronic acid or cross-linking reagent.

Hydrogels

The invention hydrogels are usually obtained by the reaction described above is modified hyaluronic acid or other suitable polymer with cross-linking reagent (also described above) under conditions which are optimal for gel formation. Typically, for optimal response correct, along with the reaction conditions, the relative amounts of reactants and reactive groups. The gel formation is carried out in mild and controlled conditions. Preferably, the resulting hydrogel contained in the aggregate less than twenty percent of unreacted functional groups contained in the original modified hyaluronic acid and cross-linking reagent, more preferably, 5% or less of unreacted functional groups contained in the original modified hyaluronic acid and cross-linking reagent, or ideally, almost undetectable amount of unreacted functional groups, such as unreacted vinylsulfonate or tirinya group. Such low levels of unreacted functional groups in the resulting gel are preferred from the viewpoint of obtaining a gel having a low anti-inflammatory properties when introduced in vivo, for example, in the joint. In a specific embodiment, the hydrogel obtained by the reaction of (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid with tilenum cross-linking reagent, contains less than ten percent of unreacted tylnej and vinylsulfonic groups. The number of unreacted functional groups is controlled by careful continuous monitoring of the reaction conditions, by adjusting the ratio of reactants, and having information about the degree of modification of the original hyaluronic acid.

The resulting hydrogels can usually contain the mass percentage of the polymer in water (POLY/HOH) in the range from about 0.5 to 5.0 percent or even more. In one or more embodiments of the implementation, as illustrations of mass p is Acento polymer in water to obtain hydrogel choose to 0.5, 1,0, 1,5, 2,0, 2,5, 3,0, 3,5, 4,0, 4,5 and 5 percent.

The education offered by the invention is lightly crosslinked hydrogels is due, at least partially, a low degree of modification of the original modified hyaluronic acid. For example, the degree of modification of hyaluronic acid in the reaction with diphenylsulfone can be controlled by appropriate adjustment of the reaction time, as shown in Example 6. For example, in order to maintain the degree of modification below about 20%, typically used in the reaction time for the formation of 2-(vinylsulphonyl)ethoxy)1-20%hyaluronic acid at ambient conditions (e.g., 20-25°C) for about 3 minutes. The reaction is preferably carried out with a molar excess of diphenylsulfone or other appropriate reagent modifications, such as difunctional or multifunctional acrylate or methacrylate reagent. As can be seen from the results in Example 6, and as you might expect, the shorter the reaction time to a lower degree of modification of the original polymer, for example, hyaluronic acid. For example, if the environmental conditions are very short reaction time, i.e. of the order of several seconds, resulted in modified vinylsulfonate hyaluronic acid having about 4% of replacement on venilale the new group, and a reaction time of one minute resulted in hyaluronic acid substitution 8% on vinylsulfonate group. To illustrate the influence of the reaction time and conditions on the achieved degree of substitution in the resulting polymer, see Table 1. In one embodiment, the reaction conditions are adjusted to obtain substituted vinylsulfonate hyaluronic acid, having from about 1% to about 10% substitution. In a related embodiment, the modification reaction is carried out at ambient conditions for approximately 10 seconds to approximately 120 seconds. The modification reaction, for example, the reaction of hyaluronic acid and diphenylsulfone, can be carried out under alkaline conditions, for example, in an aqueous alkali solution such as aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, or using any other suitable bases which are soluble in water, and then the reaction can be stopped by adding acid, such as hydrochloric acid, sulfuric acid, phosphoric acid, or other acid. Usually, the acid is added at a time and in a quantity sufficient to adjust the pH in the range from about 4 to 6.5 or so, when you want to stop the reaction in order to achieve the required degree option is the ratification of the functional groups of the original polymer. Then the product may optionally be subjected to purification, for example by dialysis, and need not be dried, for example by lyophilization.

The composition of the precursor hydrogel then slightly sew, optionally, in the presence of a crosslinking reagent, if necessary. For example, modified by vinylsulfonate hyaluronic acid, such as described above 2-(vinylsulphonyl)ethoxy)1-20%hyaluronic acid, or 2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid, is reacted with a suitable cross-linking reagent, such as functionalized by thiol PEG, such as described above PEG-dithiol, or other suitable cross-linking reagent under the reaction conditions, leading to the formation of cross-linked hydrogel. In a preferred embodiment, the crosslinking reaction is carried out in aqueous solution, for example, under physiological conditions. In one embodiment, the reaction is performed in a physiological solution. See, e.g., Examples 2, 3, 4 and 5. Volumetric relationships of the reactants can be adjusted according to the desired properties of the resulting hydrogel, and they depend on the concentrations of reagents, specific molecular mass and structure of the reagents, and other similar factors. For example, illustrations of the relative molar relations functional groups for cross-linking reagent is, when, for example, an example of a functional group is vinylsulfonic contained in the modified vinylsulfonate hyaluronic acid, and the relative amount of crosslinking reagent refers to a crosslinking reagent, for example, the molecule cross-linking reagent, and not to the number of reactive groups, such as tirinya group contained in the molecule cross-linking reagent, such as PEG-dithiol, include the following relations: approximately 1 to 2.5, or about 1.25 to 2.0, or from about 1.3 to approximately 1.8. Alternatively, cross-linking reagent may be added as solids to a solution of the modified hyaluronic acid. When you want a sterile dosage form, a crosslinking reagent sterilized before adding it, for example, by using electron beam processing. The crosslinking reaction is usually carried out under mild reaction conditions, for example, at temperatures in the range from about 20°C to about 45°C, for example, when any one of the following temperatures: 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C. After mixing or reaction of the modified hyaluronic acid and cross-linking reagent, and any other optional reactants obtained composers who s usually subjected to interaction, for example, in thermostat, in a period of time sufficient for the gel formation. Depending on the reaction temperature, the reagents are usually subjected to interaction during the period of time from about 8 to about 36 hours, or from about 10 to about 24 hours, or from about 12 to about 18 hours.

The crosslinking reaction may be carried out under sterile conditions, that is, using sterile reagents and under sterile conditions, typically described in the accompanying examples, to obtain a sterile hydrogel. For example, all components of the solution can be sterile filtered prior to the reaction, which allows to obtain a sterile composition.

Additional examples of slightly cross-linked hydrogels receive, for example, by stitching materials of thiol-modified hyaluronic acid, such as Carb-S™. Material Carb-S™ produced by karboksimetilirovaniya hyaluronic acid followed by reaction with 3,3 dithiobis(propionic hydrazide), DTPH, in the presence of reagent combinations, and then by restoring disulfide groups by using such a reagent as dithiothreitol. See, for example, patent document U.S. Patent Publication No. US2008-002595. Additional materials from thiol-modified hyaluronic acid described in the patent document is NTE U. S. Patent Publication No. US2009-0105093; illustrative material described in the above publication is hyaluronic acid, derivatization by reaction with diastereomer the hydrazide. The hydrogel may also be obtained from the thiol-modified hyaluronic acid, described in U.S. Patent No. 6884788. In a preferred embodiment, the above-mentioned materials of the thiol-modified hyaluronic acid obtained using the described methods of synthesis, except that the degree of modification of the source material from hyaluronic acid is low, resulting in less than about 20% or, more preferably, less than approximately 10% of the hydroxyl groups of hyaluronic acid are subjected to chemical modification. These materials derivateservlet the thiol hyaluronic acid can be lightly stitched because of the ability of thiols to spontaneous interaction. Alternatively, lightly crosslinked hydrogel can be obtained by reaction with a crosslinking reagent, such as PEG-acrylate.

In one or more specific variants of implementation, the composition is crosslinked hydrogel contains an active agent. Preferred classes of bioactive agents include steroids, growth factors, anti-proliferative funds and antibiotics. One particularly pre is respectful class of active funds for inclusion in the hydrogels of the present invention are corticosteroids. Illustration of corticosteroids include, but are not limited to, the following corticosteroids: triamcinolone, salt, triamcinolone, such as triamcinolone acetonide, hexacetonide triamcinolone, benetone triamcinolone, puretone triamcinolone and diacetate triamcinolone and other corticosteroids, and methylprednisolone. Usually, the resulting hydrogel contains approximately from 0.01% by weight to about 20% by weight of bioactive funds, depending on its activity. Illustrative amounts of the bioactive agent contained in the hydrogel (from the total mass of the wet hydrogel) are approximately 10% to approximately 20% by weight, for example, for the less active biologically active agent, and from about 0.01% to about 10% by weight, or from about 0.01% to about 5%, or from about 0.01% to about 3%, or from about 0.1 to about 2% of the bioactive agent, or even from about 0.1 to about 1% of the bioactive agent, for example, more active bioactive agent, such as triamcinolone acetonide. In specific embodiments, the implementation, the hydrogel used for the delivery of poorly water-soluble bioactive agent by incorporating such a bioactive agent in the hydrogel.

Preferably, the hydrogels of the present izopet the deposits received and mild reaction conditions, and in the absence of a polymerization initiator. In addition, sufficient gelation occurs and in the absence of an external energy source. For example, the gelation reaction can be carried out at a temperature in the range from about 20°C to 45°C and in the absence of initiators and accelerators. In addition, gelation, that is, the formation of the hydrogel occurs without allocating any side Monomeric chemical products. Therefore, the invention hydrogels contain the minimum number of additives or impurities, which could in principle induce proinflammatory response when introduced in vivo.

Sterile hydrogels can be obtained under sterile conditions, for example, by placing the aqueous solutions of the modified hyaluronic acid and cross-linking reagent in a separate sterile syringe and/or centrifuge tube, and then thoroughly mixing. Usually, mix the reagents (i.e., modified hyaluronic acid, and a crosslinking reagent) is placed in a thermostat set at the appropriate temperature (for example, in the range from about 20°C to 45°C), as long as the material does not form a gel. See, for example, Example 2, 18, 21, 22, 23, 24, 25, 27, 28, 29 and 30 for typical methods of preparation of the compositions of the hydrogels, including examples surround the city, the making of the reactants.

Additional unmodified hyaluronic acid, usually in the form of an aqueous solution or mixture may optionally be added to the composition of the precursor gel to gel formation or after formation of the gel (for example, in a suspension gel), to obtain a composition comprising particles of crosslinked hydrogel in an aqueous solution of hyaluronic acid. See, e.g., Example 8. Srednekislye molecular weight hyaluronic acid (namely, unmodified hyaluronic acid) in the solution is typically in the range from about 750,000 people to approximately 1200000 daltons or even higher. The preferred aqueous solution is a saline solution of hyaluronic acid, which approximate aqueous solutions of hyaluronic acid that is added to the hydrogel, have a concentration in the range from about 0.3% to about 4%, or from about 0.5% to about 2% by weight. One typical composition includes the following relative amounts of components: 4 ml of suspension gel ((2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid/PEG-dithiol) with 2 ml of hyaluronic acid at a concentration of 20 mg/ml is Particularly preferred composition comprises 4 ml of suspension gel ((2-(vinylsulphonyl)ethoxy)4%hyaluronic acid/PEG-dithiol) with 2 ml of hyaluronic acid when the concentration is AI 20 mg/ml Usually, the final content of hyaluronic acid in the resulting swollen gel is in the range from about 0.05 to 5 percent (from 0.5 mg/ml to 50 mg/ml). Preferably, the final content of hyaluronic acid in the resulting swollen gel was approximately from 0.1 to 3 percent, or from about 0.1 to 1 percent, or from about 0.5 to 0.8%. Illustrative of the final content of hyaluronic acid in the resulting swollen gel may, for example, to match any of the following values percent 0,1 , 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1,0, 2,0, 3,0, 4,0 and 5.0. For example, typical relative quantity (mass relations) of hyaluronic acid to the particles of crosslinked (for example, (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid/PEG-dithiol) hydrogel in the resulting compositions will typically range from about 10:1, or from about 5:1, or from about 3:1, or from about 1:1. The resulting composition may also optionally contain one or more surfactants. Illustration of surfactants include sodium carboxymethylcellulose, Polysorbate 80, tween 80, polyethylene glycol (e.g., PEG 3350), and other similar substances.

Optionally, the bioactive agent may be added to the reaction mixture before stapling or, alternatively, added to the crosslinked gel after its formation. Examples 9-16 illustrate the obtaining of the hydrogel, as well as the inclusion and subsequent slow release of bioactive agent, triamcinolone acetonide, typical compositions of the hydrogel. Alternatively, in the tested hydrogels can be enabled living cells, such as stem cells, parenchymal stem cells extracted from the blood cells and bone marrow cells.

In the case of the tested hydrogels with incorporated bioactive agent, or without it, the hydrogel may be dispersed in a solution of one or more of the above-described polyanionic polysaccharides (PAS). Non-limiting examples of polyanionic polysaccharides in addition to hyaluronic acid (ha) include, for example, carboxymethylcellulose (CMC), carboxymethylamino (KMA), chondroitin-4-sulfate, chondroitin-6-sulfate, sulfate of dermatan, leatherette-6-sulfate, heparin sulfate, heparin, keratin sulfate and their derivatives, and combinations thereof. Such polymers are known in the art and described, for example, in U.S. Patent No. 6056970. Other polymer solutions, in which the tested hydrogels can be dispersed include fibrin, fibrinogen, starch, polyaminoamide; peptides, proteins, gelatin, collagen, and polyethylene glycol. For dispersion of particles of the tested hydrogel can be used a solution containing one or more of the combinations mentioned in the above polymers. The polymer solutions can be prepared in the concentration range of at least 0.1 mg/ml to a maximum solubility in water or 0.9% saline solution. As has been previously described, one preferred polymer is hyaluronic acid with a molecular weight of from 500,000 to 3 million in the concentration range from about 10 mg/ml to about 25 mg/ml of the Combination of the polymer solution and hydrogel can be prepared under sterile conditions, which is why the final packaged combination is sterile.

As described in Example 8, the tested hydrogels can be mixed in different ratios with a solution of the selected polymer. Volume relations by mixing the tested hydrogel and polymer solution can include, but are not limited to, approximately 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. The preferred volumetric relations by mixing the tested hydrogel and solution of the polymer is about 3:1, 2:1 and 1:1.

The pH of the dispersions of the tested hydrogels and dispersions combinations of the tested hydrogel/polymer solution can be changed by adding buffers, acids and bases. The preferred range of pH for dispersions of the tested hydrogels and dispersions combinations of the tested hydrogel/polymer solution is is roughly 5-8, and more preferably, approximately 6-7,6.

Ionic strength of the dispersion of the tested hydrogels and dispersions combinations of the tested hydrogel/polymer solution can be changed by adding salts. One preferred salt used to change the ionic strength of the dispersion of the tested hydrogels and dispersions combinations of the tested hydrogel/polymer solution is sodium chloride. The preferred final ionic strength of the dispersion of the tested hydrogels and dispersions combinations of the tested hydrogel/polymer solution are chosen so that the variance of the tested hydrogels and dispersion combinations of the tested hydrogel/polymer solution was approximately isotonic.

In the dispersion of the tested hydrogels and dispersion combinations of the tested hydrogel/polymer solution can also be added pharmaceutically acceptable preservatives. They may include such compounds as sodium benzoate or benzyl alcohol.

The variance of the tested hydrogels and dispersion combinations of the tested hydrogel/polymer solution can, in particular variants of implementation, to be packaged in a syringe. The syringe may be made of plastic (such as polypropylene, polycarbonate, polystyrene or glass or any other pharmaceutically acceptable material. The amount of dispersion of the tested hydrogel and dispersions combinations of the tested hydrogel/polymer solution, contained in the syringe, may be approximately from 0.5 ml to 20 ml, with a preferred volume 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml and 7 ml

The resulting hydrogel material may be processed into particles with a size in the range of approximately 0.10 to 3.0 millimeters (see, for example, Examples 3 and 4), or may be in the form of a water suspension of the gel. For example, a gel-like material can be broken down into pieces, mixed with saline solution and subjected to swelling. Then from the gel-like material can be obtained particles of the appropriate size by extrusion through a sieve with an appropriate cell size, for example, approximately from 0.10 to 3.0 millimeters. The resulting particles when placed in an aqueous environment to form a suspension of the gel. In one embodiment, the gel is Packed in a syringe suitable for use with needle size 18-21, resulting hydrogel can be injected into the intra-articular space. Usually, the amount of the composition of the hydrogel, in injectable intra-articular space of a subject is from about 0.5 to about 8 ml, preferably about 3 to 6 ml, or even approximately 4-6 ml

As shown in the accompanying Examples, the hydrogels can be obtained in the form of sterile compositions.

Described above and in the Examples, the hydrogels may be offered in an airtight container, such as a syringe (which can be closed, not necessarily by using a cap with a hole). The syringe may then be placed in the packaging, such as foil bag, which is then sealed. The package can be sealed under vacuum, sealed in an atmosphere of inert gas, such as nitrogen or argon, or sealed after one or more cycles of evacuation/filling gas, where the gas for filling an inert gas, such as nitrogen or argon. In case the package is sealed with one or more cycles of evacuation/filling gas, the cycle can be adjusted so that the package was eventually sealed either under vacuum or with an inert gas. The package may optionally contain a desiccant and/or an oxygen scavenger.

Application

Described in the invention compositions of the gel have an advantage due to reduced undesirable side effects on cartilage compared with the produce industry viscoelastic supplements and options for implementation, in which the hydrogel further includes a bioactive agent, characterized by reduced undesirable side effects on cartilage compared with the introduction of an equivalent amount of the active agent without its inclusion in the hydrogel. The invention comp the positions of the gel is characterized by, along with other positive characteristics, extremely low proinflammatory properties, illustrated in Example 17 and in Figures 4-8.

Described in the invention polymer compositions of hyaluronic acid can be used in injectable or implantable dosage forms, for use, for example, in embryonic development, tissue formation, wound healing, angiogenesis, and oncogenesis. See, D. D. Allison and K. J. Grande-Allen, Tissue Engineering, Vol. 12, Number 8, 2131-2140 (2006), G. D. Prestwich et al, Tissue Engineering, Vol. 12, Number 8, 2171-2180 (2006), G. D. Prestwich et al, Tissue Engineering, Vol. 12, Number 12, 3405-3416 (2006). The hydrogel composition comprising a corticosteroid, such as triamcinolone acetonide, is used to facilitate the test subject's pain. Injection of a therapeutically effective amount of the composition of the hydrogel in the intra-articular space of a joint can provide, for example, long-lasting relief experienced by the subject of pain in the joint. Ideally, if after the injection of the subject from the very beginning experiences at an appreciable degree of pain relief in the interval from one hour to one week, or more preferably, from about one hour to about one day after injection. Usually, the injection of the hydrogel causes some degree of pain relief during the period from approximately three to nine months after inye the tion. Depending on a specific subject and subjected to the treatment condition, the amount of therapeutically effective doses of the hydrogel is typically from about 0.5 ml to 20 ml, and the sample volumes include 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml and 7 ml

For example, the invention of the composition of the hydrogel, optionally containing one or more bioactive agents, can be used as adhesive compositions, for example, as tissue adhesives and sealants that can be used in various applications, including the prevention of bleeding, closing open wounds, and other biomedical applications. These compositions can be used, for example, when sewing surgically cut or torn as a result of injury to tissues, to stop bleeding, such as bleeding from wounds, to prevent restenosis or coagulation of blood for delivery of medicines; to eliminate dimples on the skin and to promote healing and restoration of living tissue. By the invention the polymer composition on the basis of hyaluronic acid can be used to add or induction and regeneration of damaged organs or tissues of a mammal, such as man. Composition after introduction or inclusion of the subject Roslavets settled or resolved, or, alternatively, remains in the body of a subject (e.g. a mammal) without negative for his actions.

The subjects of the composition can be used as tissue fillers, dermal fillers, means for increasing the volume and invagination means, and also as a means to resolve defects/injuries of cartilage and means of enhancing the recovery and/or growth of bone.

The subjects of the composition can also be used in the treatment of osteoarthritis or rheumatoid arthritis, or other inflammatory arthritis, such as podagra or illness deposition of crystals of calcium pyrophosphate (for example, by injection into the intra-articular space of a joint), or to reduce or prevent adhesions, which may be formed after surgical operations. It was found that the subjects of the composition can be applied to reduce damage to the cartilage injections of corticosteroid inclusion of corticosteroid in the invention, the hydrogel material.

As a specific method of evaluation of the above positive effects for the case when the hydrogel contains it contained corticosteroid and method reduces cartilage damage compared to the damage of the cartilage with the introduction of an equivalent amount of corticosteroid is without its inclusion in the hydrogel, this reduced cartilage damage is characterized using the total score on the assessment system Mancina through 28 days after injection in the joint in experimental models on the goats. See Example 34 to describe the amount of points on the evaluation Mancina. In addition, can be used additional indicators for assessing reduce the damage of cartilage; these parameters and the corresponding data is also shown in Example 34.

Some of the benefits associated with capture/imprisonment particles steroid within the proposed invention the hydrogel include one or more of the following. For example, the capture particles of steroids inside the hydrogels of the present invention can prevent the direct contact of the greater part of the particles of the steroid with the tissues of the joint. Moreover, the capture particles of steroids inside the hydrogels of the present invention makes it possible to maximize the local concentration of the steroid in the joint, and at the same time to minimize its systemic concentration. In addition, the capture particles steroid dosage forms hydrogels of the present invention prevents premature departure particles steroid of the joint. And finally, as a result of capture of steroid inside the hydrogel, therapeutic efficacy of steroid is achieved at a lower total dose than in the case of the ez capture steroid inside the hydrogel, consequently minimized unsolicited local and systemic side effects. See, for example, Examples 14-16, together with Figures 2, 3 and 4, showing a linear dependence of release of drug from the example of the composition of the hydrogel in a controlled and slow motion release over time.

In the case of compositions based hydrogels comprising the bioactive agent, such compositions can be used as a delivery system for drugs for treatment along with other conditions such as osteoarthritis, sinusitis, allergic rhinitis and chronic rhinosinusitis. Such compositions can also be used as fillers for skin remedy defects/damage of cartilage and means to speed up the recovery and/or growth of bone.

Further, the present application the invention is described with specific options for implementation, which are assumed not to limit the scope of the invention. And on the contrary, the present application for the invention covers all alternatives, modifications and equivalents be included in the scope of the claims. Thus, for the purpose of illustration, specific embodiments and to facilitate understanding of the methods and conceptual aspects of the invention will be further described Khujand is the implementation of the present invention.

EXAMPLES

The following examples are given in order to present the usual experts in this field a complete disclosure and description of how the invention compounds, compositions and methods receive, carry out and evaluate, and it is assumed that these examples are only illustrations. Therefore, the examples in no way limit the scope of the invention. There are numerous variations and combinations of reaction conditions, for example, concentrations of components, suitable solvents, mixtures of solvents, temperatures, pressures and other reaction parameters and conditions, which can be used to optimize product characteristics such as purity, yield, and other such settings. It is believed that they are also included in the scope of the present invention.

EXAMPLE 1

SYNTHESIS DERIVATESERVLET VINYLSULFONATE HYALURONIC ACID (ha-sun) - LOW DEGREE of MODIFICATION

In a round bottom flask with a volume of 1 l was loaded with 5 g of hyaluronic acid (ha) [9,4×104SDRs (3% in water)]. Added to GK 500 ml of sterile filtered water. The flask was placed in a rotary evaporator, the speed of which was maintained in the range of 20-100 rpm Flask with a solution rotated until then, until it dissolved all Ledger (approximately 16-18 hours). Then the races is the thief ON (10 mg/ml) was transferred to a glass laboratory beaker with a volume of 1 L. Was placed in a mortar mixer, which was connected at the top with a drive device for mixing, and set the stirring speed, which provides intensive mixing of the solution. Added to the mix Ledger solution 333 ml of 0.25 N NaOH solution (83,2 ml of 1N NaOH added to 249,8 ml deionized water). After approximately 1 minute, stir the solution was quickly added to 150 ml of diphenylsulfone (18 ml diphenylsulfone dissolved in 132 ml of deionized water). After 15 seconds (from the time of completion of addition of solution of diphenylsulfone), adjust the pH value of the solution in the interval from 5 to 6 by quickly adding approximately 14 ml of 6N HCl. Then the reaction solution was subjected to dialysis using a flow-through filter along the stream system (Spectra/Por™, the cartridge P/N M6-100S-301-01A). The total volume was 11 times the volume of the original solution. After the stage of purification, the solution was concentrated to approximately 14-20 mg/ml Functionalized by vinylsulfonate GK (GK-SU) was removed from the system by filtering along the stream and placed in a plastic container, which was then closed with a screw top lid. Selected sample SC-SU, was frozen at -80°C and then liofilizirovanny. The dried sample was sent for analysis using the1H-NMR.

p> Definition percent replacement by vinylsulfonate for the Ledger-SU

Approximately 15-17 mg of dried sample was loaded into a tared test tube with a volume of 2 ml Sample resuspendable 1.5 ml of D2O. the Sample was transferred into a vial for NMR spectroscopy. Filmed1H-NMR spectrum (256 scans) of the sample and printed it with specific peaks in areas with a 6.3 to 6.5 ppm (2 peak 2 CH2=protons from vinylsulfonate residue) and 1.5-2.5 ppm (singlet 3 CH3-protons from the N-acetyl group of the civil code), which are integrated. The percentage of modification was calculated as follows:

% modification = 150×(the integral of the peaks of vinylsulfonic)/(integral peak ndimethylacetamide).

1H-NMR spectrum (FIG. 1) showed that GK had a degree of substitution of vinylsulfonate approximately 4%, based on the integration of the peaks of vinylsulfonic relative to the methyl group of ndimethylacetamide hyaluronic acid.

Sample GC-SU used to determine the mass in the dry state, which was used to determine the specific concentration of GC-SC solution. The concentration of SC-SU was 18 mg/ml

EXAMPLE 2

The SYNTHESIS GEL, PREPARED FROM a MODIFIED VINYLSULFONATE HYALURONIC ACID (ha-sun) AND PEG-DITHIOL

Solution SC-SU, prepared as described in Example 1 was diluted with deionized water to a concentration of 14 mg/ml was Placed ml solution SC-SU in sterile syringe with a volume of 20 ml. Solution SC-SU was filtered through a syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml to Prepare a solution of 50 mg/ml PEG-(SH)2[company Laysan Bio Inc., the position in the directory # SH-PEG-SH-3400-1g] by dissolving 40,1 mg PEG-(SH)2in 0,802 ml of deionized water. A solution of PEG-(SH)2was transferred into a sterile syringe with a volume of 1 ml and filtered through a syringe filter with a pore size of 0.2 μm. Transferred to 10 ml of sterile filtered Ledger-SU in a sterile centrifuge tube with a volume of 50 ml was Added to a solution of SC-SC 250 ál of a 0.5 M solution of sodium phosphate (filtered through a syringe filter with a pore size of 0.2 μm). The resulting solution was thoroughly mixed. To a solution of SC-SU was added 380 µl [19 mg PEG-(SH)2] sterile 50 mg/ml PEG-(SH)2. The resulting solution was thoroughly mixed. To ensure sterility, above the stage had been Boxing for conducting biological research. Then a solution of ha-VS/PEG-(SH)2was placed in a thermostat with a temperature of 37°C for at least 16 hours to activate the gel formation. At least, after 16 hours, the solution GK-VS/PEG-(SH)2sewn together with the formation of the gel. Then the gel-like material was removed from thermostat.

EXAMPLE 3

PREPARATION of SUSPENSION GEL of ha-VS/PEG-(SH)2- SINGLE EXTRUSION

The gel is To-VS/PEG-(SH) 2from Example 2 was mechanically crushed into pieces with a glass rod. Transferred the gel in a sterile syringe with a volume of 60 ml, which was closed with the cap. To the gel was added 40 ml of 0.9% sterile NaCl solution. Put the piston in the cylinder of the syringe, and the syringe turned. Removed from the syringe cap for dropping excess pressure, and then the cap was worn. The syringe turned several times to ensure good mixing of saline and gel pieces. Spent the swelling of the gel during the night (at least for 16 hours).

With a punch to the skin with a diameter of 23 mm were cut from polyester mesh (company McMaster Carr, # catalog 9218T13, the hole dimensions: 20,3 x 20,3, the size of the squares/rectangles: 0,0331", the nominal value in microns: 840 microns, the proportion living section: 46, the diameter of the thread: 0,0157") disc diameter 23 mm Disk was inserted into the holder syringe filter 25 mm diameter (Cole Palmer, # catalog EW-29550-42), and the filter holder was closed. The filter holder containing the grid, sterilized in an autoclave. With the syringe removed the cap and syringe filter containing the grid, combined with a syringe. The gel was extrudible through the mesh into a sterile centrifuge tube with a volume of 50 ml. Centrifuge tube was closed with a screw top lid. The resulting product which was slightly viscous suspension of particles, from which particles are practically were not deposited, and usually remained in suspended form. Above the stage was implemented in Boxing for conducting biological research.

EXAMPLE 4

PREPARATION of SUSPENSION GEL of ha-VS/PEG-(SH)2- DOUBLE EXTRUSION

Gel GK-VS/PEG-(SH)2from Example 2 was mechanically crushed into pieces with a glass rod. The gel was transferred into a sterile syringe with a volume of 60 ml, which was closed with the cap. To the gel was added 40 ml of 0.9% sterile NaCl solution. Put the piston in the cylinder of the syringe, and the syringe turned. Removed from the syringe cap for dropping excess pressure, and then the cap was worn. The syringe turned several times to ensure good mixing of saline and gel pieces. Spent the swelling of the gel during the night (at least for 16 hours).

With a punch to the skin with a diameter of 23 mm were cut from polyester mesh (company McMaster Carr, # catalog 9218T13, the hole dimensions: 20,3 x 20,3, the size of the squares/rectangles: 0,0331", the nominal value in microns: 840 microns, the proportion living section: 46, the diameter of the thread: 0,0157") disc diameter 23 mm Disk was inserted into the holder syringe filter 25 mm diameter (Cole Palmer, # catalog EW-29550-42), and the filter holder was closed. The filter holder containing the CE is ku, sterilized in the autoclave. With the syringe removed the cap and syringe filter containing the grid, combined with a syringe. The gel was extrudible through the mesh into a sterile centrifuge tube with a volume of 50 ml. Extruded gel was then placed in a sterile syringe with a volume of 60 ml, and the syringe was connected with syringe filter containing a grid. The gel was extrudible through the mesh into a sterile centrifuge tube with a volume of 50 ml. Centrifuge tube was closed with a screw top lid. Above the stage was implemented in Boxing for conducting biological research.

EXAMPLE 5

PREPARATION of SYRINGES LOADED SUSPENSION GEL of ha-VS/PEG-(SH)2

In a sterile glass syringe (B&D) a volume of 10 ml, which was equipped cap, transfer 5 ml of the prepared suspension gel of ha-VS/PEG-(SH)2(from Example 3 or Example 4). In the upper part of the syringe was inserted a sterile tube. The piston rod is put in the tube. The syringe turned, and as soon as the suspension of the gel was achieved tube, cap the syringe slightly weakened and pushed the piston up until the syringe was not removed a large part of the air. The syringe was wearing a cap. Above the stage was implemented in Boxing for conducting biological research.

EXAMPLE 6

SYNTHESIS DERIVATESERVLET VINYLSULFONATE HYALURONIC IS SLOTY

To prepare the composition of GC-SU, with different degrees of substitution, using the method described in Example 1, except that the increased time of reaction. By increasing the reaction time, are given a higher degree vinylsulfonate replacement. The results of these reactions are given in the table below:

Table 1
The REACTION TIMEThe DEGREE of SUBSTITUTION FOR the Ledger MODIFIED USING SU
15 seconds4%
1 minute8%
3 minutes20%
5 minutes26%
25 minutes29%

EXAMPLE 7

The SYNTHESIS GEL of ha-VS/PEG-(SH)2

Samples GC-SU, with varying degrees vinylsulfonate substitution (Example 6) was used to prepare gels of ha-VS/PEG-(SH)2using the method and proportions of the reagents described in Example 2. Each of the source materials was formed into a gel by reaction with PEG-dithiol.

EXAMPLE 8

PREPARATION of SUSPENSION GEL of ha-VS/PEG-(SH) With HYALURONIC ACID

In a round bottom flask of 250 ml was loaded with 2 g of hyaluronic acid [9,4×104SDRs (3% in water)]. Added to the hyaluronic acid in the flask 100 ml of sterile physiological solution. The flask was placed in a rotary evaporator (Buchi) and rotated at a speed of 50 rpm, at least for 16 hours to obtain a 2% solution of hyaluronic acid. The subsequent series of stages carried out in Boxing for conducting biological research. A solution of hyaluronic acid was filtered through a sterile filter with a pore size of 0.2 μm. Using suspension gel of ha-VS/PEG-(SH)2(prepared in Example 3 or 4), to prepare a number of compositions for which the prepared suspension gel of ha-VS/PEG-(SH)2mixed with hyaluronic acid. Volumes of a solution of hyaluronic acid and suspension gel of ha-VS/PEG - (SH)2used to prepare the compositions shown in the table below:

Table 2
TRACKThe AMOUNT of HYALURONIC ACID (ML)The AMOUNT of SUSPENSION GEL of ha-VS/PEG-(SH)2(ML)
115 (suspension after a single extrusion)
224 (suspension after a single extrusion)
333 (suspension after a single extrusion)
442 (suspension after a single extrusion)
551 (suspension after a single extrusion)
815 (suspension after two extrusion)
724 (suspension after two extrusion)
833 (suspension after two extrusion)
942 (suspension after two extrusion)
1051 (suspension after two extrusion)

The indicated volumes of a solution of hyaluronic acid and suspension gel of ha-VS/PEG-(SH)2shown in the table above were added to sterile centrifuge tube about the reception 15 ml. The tube was closed with a lid, and the test tube is turned up and down, until, until well mixed components. Each composition then was transferred to a glass syringe with a volume of 10 ml, which has a cap, then insert the piston and remove excess air. Then the syringe was put on the cap.

EXAMPLE 9

The SYNTHESIS GEL of ha-VS-PEG-(SH)2CONTAINING ACETONIDE TRIAMCINOLONE

Solution SC-SU, prepared as described in Example 1 was diluted with deionized water to a concentration of 14 mg/ml was Placed 11 ml solution GC-SC in sterile syringe with a volume of 20 ml Solution GC-SU was filtered through a syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml to Prepare a solution of 50 mg/ml PEG-(SH)2by dissolving 40,1 mg PEG-(SH)2in 0,802 ml of deionized water. A solution of PEG-(SH)2was transferred into a sterile syringe with a volume of 1 ml and filtered through a sterile syringe filter with a pore size of 0.2 μm. Transferred to 10 ml of sterile filtered Ledger-SU in a sterile centrifuge tube with a volume of 50 ml was Added to a solution of SC-SC 100 mg of triamcinolone acetonide (Spectrum Chemicals, pharmaceutical purity, finely chopped). Closed centrifuge tube cap, and the solution turned it waver, down, up until the triamcinolone acetonide was not homogen is mixed with SC-SU. Was added to a solution of SC-SC 250 ál of a 0.5 M solution of sodium phosphate (filtered through a syringe filter with a pore size of 0.2 μm). The resulting solution was thoroughly mixed. To a solution of SC-SU was added 380 ál of sterile 50 mg/ml PEG-(SH)2. The resulting solution was thoroughly mixed. Above the stage was implemented in Boxing for conducting biological research. Then a solution of ha-VS/PEG-(SH)2was placed in a thermostat with a temperature of 37°C for at least 16 hours to activate the gel formation. At this stage, a solution of ha-VS/PEG - (SH)2sewn together with the formation of the gel. Then the gel-like material was removed from thermostat. The resulting gel contains about 0.2% triamcinolone acetonide.

The above procedure was conducted in the same manner as above, except that the solution Ledger-SU was added 20 mg of triamcinolone acetonide (Spectrum Chemicals, pharmaceutical purity, finely chopped).

EXAMPLE 10

PREPARATION of SUSPENSION GEL of ha-VS-PEG-(SH)2CONTAINING TRIAMCINOLONE ACETONIDE: a SINGLE EXTRUSION

Containing triamcinolone acetonide gel GK-VS/PEG-(SH)2(Example 9) was mechanically crushed into pieces with a glass rod. Transferred the gel in a sterile syringe with a volume of 60 ml, which was closed with the cap. To the gel was added 40 ml of 0.9% sterile is on NaCl. Put the piston in the cylinder of the syringe, and the syringe turned. Removed from the syringe cap for dropping excess pressure, and then the cap was worn. The syringe turned several times to ensure good mixing of saline and gel pieces. Spent the swelling of the gel during the night (at least for 16 hours).

With a punch to the skin with a diameter of 23 mm were cut from polyester mesh (company McMaster Carr, # catalog 9218T13, the hole dimensions: 20,3 x 20,3, the size of the squares/rectangles: 0,0331", the nominal value in microns: 840 microns, the proportion living section: 46, the diameter of the thread: 0,0157") disc diameter 23 mm Disk was inserted into the holder syringe filter 25 mm diameter (Cole Palmer, # catalog EW-29550-42), and the filter holder was closed. The filter holder containing the grid, sterilized in an autoclave. With the syringe removed the cap and syringe filter containing the grid, combined with a syringe. The gel was extrudible through the mesh into a sterile centrifuge tube with a volume of 50 ml. Centrifuge tube was closed with a screw top lid. Above the stage was implemented in Boxing for conducting biological research.

EXAMPLE 11

PREPARATION of SUSPENSION GEL of ha-VS-PEG-(SH)2CONTAINING TRIAMCINOLONE ACETONIDE: a DOUBLE EXTRUSION

Containing acetone is d triamcinolone gel GK-VS/PEG-(SH) 2(Example 9) was mechanically crushed into pieces with a glass rod. The gel was transferred into a sterile syringe with a volume of 60 ml, which was closed with the cap. To the gel was added 40 ml of 0.9% sterile NaCl solution. Put the piston in the cylinder of the syringe, and the syringe turned. Removed from the syringe cap for dropping excess pressure, and then the cap was worn. The syringe turned several times to ensure good mixing of saline and gel pieces. Spent the swelling of the gel during the night (at least for 16 hours).

With a punch to the skin with a diameter of 23 mm were cut from polyester mesh (company McMaster Carr, # catalog 9218T13, the hole dimensions: 20,3 x 20,3, the size of the squares/rectangles: 0,0331", the nominal value in microns: 840 microns, the proportion living section: 46, the diameter of the thread: 0,0157") disc diameter 23 mm Disk was inserted into the holder syringe filter 25 mm diameter (Cole Palmer, # catalog EW-29550-42), and the filter holder was closed. The filter holder containing the grid, sterilized in an autoclave. With the syringe removed the cap and syringe filter containing the grid, combined with a syringe. The gel was extrudible through the mesh into a sterile centrifuge tube with a volume of 50 ml. Extruded gel was then placed in a sterile syringe with a volume of 60 ml, and the syringe was connected to the syringe is passed by the filter, which contained the grid. The gel was extrudible through the mesh into a sterile centrifuge tube with a volume of 50 ml. Centrifuge tube was closed with a screw top lid. Above the stage was implemented in Boxing for conducting biological research.

EXAMPLE 12

PREPARATION of SYRINGES WITH SUSPENSION GEL CONTAINING ACETONIDE TRIAMCINOLONE

In a sterile glass syringe (B&D) a volume of 10 ml, which was equipped cap, transfer 5 ml of the prepared suspension gel of ha-VS/PEG-(SH)2containing triamcinolone acetonide (Example 10 or Example 11). In the upper part of the syringe was inserted a sterile tube. The piston rod is put in the tube. The syringe turned, and as soon as the suspension of the gel was achieved tube, cap the syringe slightly weakened and pushed the piston up until the syringe was not removed a large part of the air. The syringe tightly wore a cap. Above the stage was implemented in Boxing for conducting biological research.

EXAMPLE 13

PREPARATION of SUSPENSION GEL of ha-VS/PEG-(SH)2CONTAINING TRIAMCINOLONE ACETONIDE, WITH HYALURONIC ACID

In a round bottom flask of 250 ml was loaded with 2 g of hyaluronic acid [9,4×104SDRs (3% in water)]. Added to the hyaluronic acid in the flask 100 ml of sterile physiological solution. The flask was installed is in a rotary evaporator (Buchi) and rotated at a speed of 50 rpm, at least for 16 hours to obtain a 2% solution of hyaluronic acid. Using suspension gel of ha-VS/PEG-(SH)2containing triamcinolone acetonide (obtained in Example 10 or 11), to prepare a number of compositions for which the suspension gel of ha-VS/PEG-(SH)2containing triamcinolone acetonide, mixed with hyaluronic acid. Volumes of a solution of hyaluronic acid and suspension gel of ha-VS/PEG - (SH)2containing triamcinolone acetonide used to prepare the compositions shown in the table below:

Table 3
TRACKThe AMOUNT of HYALURONIC ACID (ML)The AMOUNT of SUSPENSION GEL of ha-VS/PEG-(SH)2CONTAINING TRIAMCINOLONE ACETONIDE (ML)
115 (suspension after a single extrusion)
224 (suspension after a single extrusion)
333 (suspension after a single extrusion)
44 2 (suspension after a single extrusion)
551 (suspension after a single extrusion)
615 (suspension after two extrusion)
724 (suspension after two extrusion)
833 (suspension after two extrusion)
942 (suspension after two extrusion)
1051 (suspension after two extrusion)

The indicated volumes of a solution of hyaluronic acid and suspension gel of ha-VS/PEG-(SH)2containing triamcinolone acetonide shown in the table above were added to sterile centrifuge tube with a volume of 15 ml test Tube was closed with a lid, and the test tube is turned up and down, until, until well mixed components. Each composition then was transferred to a glass syringe with a volume of 10 ml, which has a cap, then insert the piston and remove the excess of who the ear. Then the syringe was put on the cap. Above the stage was implemented in Boxing for conducting biological research.

EXAMPLE 14

PREPARATION of SPECIMENS FOR RESEARCH RELEASE ACETONIDE TRIAMCINOLONE

Transferred to 1.5 ml containing triamcinolone acetonide gel GK-VS/PEG-(SH)2(prepared according to Example 11) in a glass scintillation vial with a capacity of 20 ml was Added via pipette to a scintillation vial containing gel-like material, 15 ml of PBS (pH 7,4). The scintillation vial was closed with a screw cap, and the bottle was placed on vibroshaker (Barnstead International, Model M26125) in a thermostat with a temperature of 37°C.

EXAMPLE 15

The SAMPLING BUFFER IN the STUDY of the RELEASE of TRIAMCINOLONE ACETONIDE

The scintillation vial containing the gel with prisoners in it triamcinolone acetonide and PBS buffer (described in Example 14), was removed from thermostat with a temperature of 37°C at different points in time. The remaining suspension of the gel was allowed to settle to the bottom of the scintillation vial. Screw cap was removed and were taken using a serological pipette, 13 ml of PBS buffer and transferred into a plastic centrifuge tube with a volume of 50 ml was Then added via pipette 13 ml of fresh PBS buffer (pH 7,4) in a scintillation vial containing gel.

PRIMER

The ANALYSIS of the ENVIRONMENT IN WHICH it was ALLOCATED TRIAMCINOLONE ACETONIDE, high performance LIQUID CHROMATOGRAPHY

13 ml of sample buffer (Example 15) was diluted to 40 ml with a mixture of MeOH:H2O=80:20. The sample was shaken, and approximately 1 ml was transferred into a vial for HPLC. The content of triamcinolone acetonide in the sample buffer was determined under the following chromatographic conditions:

Installation HPLC: Agilent 1100 series

Column: Bond SB-C18, 5 microns, 4,6×160 mm

The column temperature: 30°C

Flow rate: 1.0 ml/min

Detection: UV at 239 nm

Run time: 8 minutes

The volume of injected sample: 50 ál

Mobile phase: 0.05%of TFA in ACN: 0.05%of TFA in H2O 56:44

The retention time of triamcinolone acetonide: ~3.3 minutes

The amount of triamcinolone acetonide in the sample buffer was determined by comparing the peak area in the concentration of triamcinolone acetonide with standard curve. Samples for calibration curve of triamcinolone acetonide was obtained by initial preparation of the solution of the acetonide triamcinolone in methanol and then a serial dilution of the initial solution using a mixture of 0.05% TFA in ACN: 0.05% of TFA in H2O 56:44. These samples were analyzed under the above chromatographic conditions, and the resulting peak area was plotted on the graphical dependence on the concentration of triamcinolone acetonide. The percentage visw is bogdania acetonide triamcinolone shown in FIG. 2, the total mass released acetonide triamcinolone shown in FIG. 3, and the number of redundant triamcinolone acetonide at the time of selection of the sample shown in FIG. 4.

Samples were collected every 24 hours from Monday to Friday; Saturday and Sunday, the sampling was not performed.

Table 4
Number selection sampleTime of sampling (days)
11
22
33
47
58
69
710
811
914
1015

As shown in Fig. 2, almost all the drug was released to the time of sampling 12. The release of drugs about what went on linear dependence in time, and it can be adjusted. It is preferable that was almost complete release of the drug, and not Vice versa, as a significant amount of drugs could remain trapped in the gel. In addition, instead of the original instant release drug, the drug is released over time in a slow and steady mode. In Fig. 3 similarly shows the cumulative release of drug, in milligrams, depending on the time of sample selection. As shown in Fig. 4, the amount of drug released from the gel was almost constant between the time of sampling, indicating a linear dependence of release of drugs in a regulated and stable in time.

EXAMPLE 17

Intra-ARTICULAR INJECTION of the SAMPLE SUSPENSION GEL of ha-VS/PEG-(SH)2

The sample suspension gel of ha-VS/PEG-(SH)2(prepared as in Example 5) were injected with intra-articular in the knee (knee) goats with a Mature skeleton together with additional test materials 2-4 were used for comparison. See also the publication D. Jackson and T. Simon, Osteoarthritis and Cartilage, Vol. 14, Issue 12, p. 1248-125, which shows additional info is the information on the used experimental model for the goats.

The test MATERIAL 1: gel GK-VS/PEG-(SH)2(Example 5)

The test MATERIAL 2: diacrylate PEG made using bestilling cross-linking reagent

The test MATERIAL 3: 4-tier functionalized with lysine PEG, which was subjected to crosslinking with the formation of a gel

Test 4 MATERIAL: gel, obtained from diacrylate PEG (the material was subjected to autoclave)

See Examples 31-33 for preparation of test materials 2-4. All injections were performed under strict sterile. All animals were anestesiologi by intravenous injection of diazepam (0.1-0.5 mg/kg) and ketamine (4,4-7.5 mg/kg). Every knee was physically examined for the knockout, range of motion, swelling, temperature, crepitation, on the trajectory of the patella, and valgus/varus violations.

All injections were performed using conventional aseptic techniques. The left and right knee were prepared for injection by vystrogana wool in their respective fields and then disinfect them with a thorough treatment with chlorhexidine. The animal was transferred to the supine position. Right knee disinfected three times by careful treatment with chlorhexidine, alternating treatment with 70% isopropyl alcohol and stained with iodine solution.

For injection is every knee used a standardized methodology. A sterile needle 21 size length of 2 inches was introduced in the intra-articular space through the anteromedial access. Injected the needle until it touches the side wall mimimally cutting the medial femoral condyle and gently took her back. Were injected with the right joint, 1.5 ml of suspension gel of ha-VS/PEG - (SH)2. The injection needle was removed, and was supported by increased pressure in the injection. Injected knee then 20 times were subjected to a full cycle on "bending".

After the injection was tested those animals that showed signs of distress and discomfort, and if necessary, they have introduced additional analgesic. All methods of treatment recorded in the relevant documentation on the study.

Injectable animals were humanely euthanized after 24±1 hours after the start of injection by intravenous diazepam 0.22 mg/kg and ketamine 10 mg/kg to achieve a state of General anesthesia. Then shot the animals were injected intragastrically lethal dose of concentrated solution of potassium chloride (KCl) as long as not stated cardiac arrest.

After collecting the knee joints were opened and conducted a visual inspection of the injected knee joints, are shown in Table 5. Photographing is not performed.

Table 5
Visual assessment and collection of samples
SampleVisual assessmentCollecting samplesPoints
Synovial fluid (the left and right knee)XXX
Left and right knee jointsXX
The left and right synoviumXX

In addition, conducted a semi-quantitative estimation of the joint with a single expert, which are shown in Table 6.

Table 6
Qualitative assessment based on visual inspection
PointsStainingHyperemiaSwelling
0NormalNoNo
1Slightly yellowLightLight
2YellowModerateModerate
3VisibleVisible

The total number of points in the visual inspection was the sum of the scores of staining, hyperemia, and edema (0-8 points). See The Fig. 6.

After collecting synovial fluid from open joints, which recorded the total volume. Visual and semiquantitative scoring was assessed viscosity, transparency and the color of the liquid, as shown in Table 7. Using hemocytometer determined the total number of leukocytes. In addition, a smear was prepared synovial fluid for the differential microscopic analysis. The remaining synovial fluid was kept frozen in cryovials with individual labels at -80°C. Smear synovial fluid was retained for possible future analysis.

Table 7
Description and scoring of synovial fluid
PointsColorTransparencyTendon
0S=STRAWC=TRANSPARENTN=NORMAL
1P=PINKH=WHITEA=ABNORMAL
2Y=YELLOW R=REDD=CLOUDYW=WATERY
3B=BLOODYT=OPAQUE

The total number of points for synovial fluid was the sum of the scores of color, transparency and tendons (0-8 points).

The results are shown in graphical form in Fig. 5-8. As can be seen in Fig. 5, an example of a gel having described the invention characteristic features, was characterized by the number of cells in the synovial fluid, which was significantly lower than the number of cells observed for the tested materials 2-4. And actually, the number of cells for the tested materials 2-4 was approximately 5-fold, 9-fold and 8-fold bol is the noise value, than that observed for the test material 1. Despite the fact that the tested materials 2-4 show the in-vitro properties (e.g., chemistry, properties of the gel, ease of administration, and so forth), indicating the possibility of their pharmaceutical applications, however, these results demonstrate the clear superiority of the test material 1 and similar materials, from the standpoint of having a much lower Pro-inflammatory properties in the study in an experimental model of goats than seemingly comparable with test materials. Surprisingly, but all other indicators point to the applicability of the other of the materials tested for use as a viscoelastic supplements and in other similar applications.

The data in Fig. 6, showing the absolute number of leukocytes in synovial fluid (absolute amount=total amount × the number of leukocytes in synovial fluid) knees injected goats, further confirm the above. Namely, based on the absolute number of leukocytes in synovial fluid, a sample of the test material 1 demonstrates remarkably low response inflammatory response in experimental models goats than the tested materials 2-4. Values for the tested materials approximately 2-4 is 4 times 12 times, and more than 9 times higher than for test material 1, which indicates a striking and significant advantage of the test material 1 when evaluating knee goats.

In Fig. 7 graphically presents the differential distribution of leukocytes in synovial fluid (average values for groups) in the injected knee goats in relation to the group subjected to processing the test material, assessed 24 hours after a 1.5 ml injection, as described in Example 17.

For each test material shows the distribution polymorphically leukocytes (PMN), lymphocytes, monocytes and eosinophils (Eos). Polymorphically leukocytes and eosinophils are important cellular participants in several types of acute and chronic inflammation. The percentage polymorphically leukocytes for test material 1 (relative to lymphocytes, monocytes and eosinophils) was significantly lower than for the other tested materials (approximately 50% compared with 70% and above for the tested materials 2-4), which is another indication of the preferred lower Pro-inflammatory properties of the sample of the test material 1 in comparison with the other tested materials.

And finally, in Fig. 8 shows the average scores for synovial fluid, synovial tissue, and the joint points of the Sina is a pressing fluid and joint tissues (table 6) for the injected knee goats for each typical test material.

Total visual assessment scores=scores for synovial fluid+the Total number of points for the joint.

The maximum number of points for synovial fluid or the total number of points for the joint 8 is, in this case the rate is 0.

The maximum number of points for total visual assessment is 16, while the norm is 0.

As shown in Fig. 8, the test material 1 has demonstrated an astonishing result. And actually, based on all of the scoring exercise conducted by the above-described visual observation, it was shown that the Tested material 1 is virtually the response, inflammatory response, with scores for synovial fluid, articular tissues, and their combination, characteristic for normal or almost normal. In contrast, typical Subjects materials 2-4 gave visual assessment as to the synovial fluid and joint, which did not reflect the normal state, and pointed to the inflammation in the knee joint due to the introduction of the test material. These results demonstrate the amazing and preferred illustrative properties of the test material 1 from the point of view of potential use for therapeutic applications in vivo.

EXAMPLE 18

SYNTHESIS of G IS LA ha-VS/PEG-(SH) 4

Solution SC-SU, prepared in Example 1 was diluted with deionized water to a concentration of 12.6 mg/ml 18 ml SC-SU is placed in a sterile syringe with a volume of 20 ml Solution GC-sun filtered through a sterile syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml 200 mg PEG - (SH)4C(CH2O(CH2CH2O)nCH2CH2SH)4, [company Laysan Bio Inc, Mw 10000, the number 4armPEG-SH-10kD-1g] (subjected to electron beam processing) add 2 ml of sterile filtered to 0.17 M solution of sodium phosphate in 1M saline solution (pH of 7.4). After dissolution, the solution of PEG-(SH)4added to a solution of SC-SU. The resulting solution was thoroughly mixed. Then allow the solution GK-VS/PEG-(SH)4to form a gel at room temperature. The gel-like material can be transformed into a suspension of the gel in the same manner as described in Examples 3 and 4. The gel can be prepared in the presence of triamcinolone acetonide using the techniques described in Examples 9, 10 and 11. Hyaluronic acid can be added to the composition of the gel using the method described in Example 8. Hyaluronic acid can be added to the composition of the gel containing triamcinolone acetonide, using methods described in Example 13.

EXAMPLE 19

SYNTHESIS CARBOXYMETHYLAMINO THE NEW ACID (CM-GC OR CARBYLAN™)

An aqueous solution of NaOH (200 ml, 45% weight/volume) was added to a beaker of 500 ml and stirred (using a magnetic stirrer) at ambient temperature. Was added to the beaker 500 ml powder hyaluronic acid (20 g) [company Novozymes, the molecular mass of 0.8-1.0 million]. After settling for 2 hours, a mixture of hyaluronic acid was transferred into a beaker 4 l with 1500 ml of isopropanol and covered with a Teflon magnetic stir bar, and then was added with stirring a solution of 20 g of Chloroacetic acid in 500 ml of isopropanol. After stirring for 1 hour at ambient temperature, the stirring was stopped and gave the opportunity for the material to settle in for about 10-20 minutes. From the mixture was removed as many as possible of the supernatant liquid. To the mixture was added to 1000 ml of distilled water. After the dissolution, adjust the pH value of the solution to approximately pH 7.0 by the addition of 6.0 N HCl. Then the solution volume was brought to 2 l with deionized water. The solution was purified by flowing through the filter along the flow (TFF) using 10 l of deionized water as a clipboard.

In addition, the structure, synthesis and study carboximetilcelulosa acid described in the patent document International Patent Publication No. 2005/056608 (Fig. 5 and Example 3), the content corresponding to the t, which is given here by reference thereto.

EXAMPLE 20

The SYNTHESIS of COMPOUNDS CARBOXIMETILCELULOSA ACID-DITHIOBIS(DEHYDRATED PROPIONIC ACID) (CM-GK-DTPH OR CARBYLAN™-S)

3,3'-Dithiobis(dehydrated propanoic acid) (DTP) was synthesized according to previously published methods (Vercruysse, K. P.; Marecak, D. M.; Marecek, J. F.; Prestwich, G. D. "Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels of hyaluronic acid", Bioconjugate Chem. (1997) 8:686-694; Shu, X. Z.; Liu, Y.; Luo, Y.; Roberts, M. C.; Prestwich, G. D. "Disulfide crosslinked hyaluronan hydrogels", Biomacromolecules (2002) 3:1304-1311). Added DTP (16.7 g, 0.07 mol) prepared above solution CarbylanTMand adjust the pH value of the solution to 4.75 by adding either HCl or NaOH solution. Then added 0,384 g of 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide (EDC) [company Sigma-Aldrich], and supported the pH value of the solution at a pH of 4.75 by addition of 6.0 N HCl under continuous stirring with a magnetic stirrer at room temperature.

After 4 hours was added 50 g of dithiothreitol (DTT) [company Biovectra], and adjusting the pH value of the solution to 8.5 by the addition of concentrated NaOH solution. Then, after stirring with a magnetic stirrer at room temperature for 12-24 hours, adjust the pH value of the reaction mixture to pH 3.0 by the addition of 6.0 N HCl. The acidified solution was purified and concentrated using flowing along flow (TFF) using 20 l of 1 mm HCl, pH 3.0. Then rest the p was concentrated to a volume of approximately 1 liter

Structure, synthesis and study of compounds carboximetilcelulosa acid - dithiobis(dehydrated propanoic acid) described in patent document International Patent Publication No. 2005/056608 (Fig. 5 and Example 4), the content of the relevant parts of which can be found here by reference thereto.

EXAMPLE 21

The SYNTHESIS GEL CM-GK-DTPH/PEG-(ACRYLATE)2

A solution of CM-GK-DTPH prepared in Example 20 was diluted with deionized water to a concentration of 17.5 mg/ml In sterile syringe with a volume of 60 ml was placed 30 ml CM-GK-DTPH. A solution of CM-GK-DTPH filtered through a sterile syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml Prepared solution of 40 mg/ml PEG-(acrylate)2[company Laysan Bio Inc., molecular weight 3400, the catalog number of an ACRL-PEG-ACRL3400-1g] by dissolving 600 mg PEG-(acrylate)2in 15 ml of a buffer of 0.2 M sodium phosphate (pH 7,4). A solution of PEG-(acrylate)2transferred into a sterile syringe with a volume of 20 ml and filtered through a sterile syringe filter with a pore size of 0.2 μm. Transfer 20 ml of sterile filtered CM-GK-DPG in a sterile centrifuge tube with a volume of 50 ml, Add 10 ml solution of PEG-(acrylate)2in a solution of CM-GK-DTPH. The resulting solution was thoroughly mixed. Then allow the solution CM-GK-DTPH/PEG-(acrylate)2to form a gel at room temperature./p>

EXAMPLE 22

SUSPENSION GEL CM-GK-DTPH/PEG-(ACRYLATE)2

Gel CM-GK-DTPH/PEG-(acrylate)2(obtained in Example 21) is transformed into a suspension of the gel using the techniques described in Examples 3 and 4, respectively.

EXAMPLE 23

GEL CM-GK-DTPH/PEG-(ACRYLATE)2CONTAINING ACETONIDE TRIAMCINOLONE

A solution of CM-GK-DTPH prepared in Example 20 was diluted with deionized water to a concentration of 14 mg/ml In sterile syringe with a volume of 20 ml placed 11 ml CM-GK-DTPH. A solution of CM-GK-DTPH filtered through a sterile syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml. Prepare a solution of 50 mg/ml PEG-(acrylate)2[company Laysan Bio Inc., molecular weight 3400, the catalog number of an ACRL-PEG-an ACRL-3400-1g] by dissolving 40,1 mg PEG-(acrylate)2in 0,802 ml of deionized water. A solution of PEG-(acrylate)2transferred into a sterile syringe with a volume of 1 ml and filtered through a sterile syringe filter with a pore size of 0.2 μm. 10 ml of sterile filtered CM-GK-DTPH transferred into a sterile centrifuge tube with a volume of 50 ml was Added to a solution of CM-GK-DPG 20 mg of triamcinolone acetonide (Spectrum Chemicals, pharmacopeias purity, fine). The centrifuge tube was closed with a lid, and a test tube with a solution turned up and down, up until acetonide triamcinolone th is ogendo not mixed with CM-GK-DTPH. To a solution of CM-GK-DTPH add 250 ál of a 0.5 M solution of sodium phosphate. The resulting solution was thoroughly mixed. To a solution of CM-GK-DTPH add 380 µl [19 mg PEG-(acrylate)2] sterile solution of 50 mg/ml PEG-(acrylate)2. The resulting solution was thoroughly mixed. A solution of CM-GK-DTPH/PEG-(acrylate)2containing triamcinolone acetonide, then placed in a thermostat with a temperature of 37°C for at least 16 hours. At this stage, a solution of CM-GK-DTPH/PEG-(acrylate)2containing triamcinolone acetonide, stitched with gel formation. Then the gel-like material was removed from thermostat.

The synthesis gel is repeated, using 33 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 375 mg, 400 mg and 500 mg of triamcinolone acetonide, respectively.

Gels were turned in suspension gel using the method described in Example 3 and 4.

EXAMPLE 24

SYNTHESIS of SUSPENSION GEL of ha-VS-PEG-(SH)2,CONTAINING ACETONIDE TRIAMCINOLONE

Gels of ha-VS-PEG-(SH)2containing triamcinolone acetonide prepared using the method described in Example 9, except that for the preparation of each gel used 33 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 375 mg, 400 mg and 500 mg of triamcinolone acetonide, respectively.

Gels were turned in suspension gel using the techniques described in Primera and 4.

EXAMPLE 25

The SYNTHESIS GEL CM-GK-DTPH/PEG-(ACRYLATE)4

A solution of CM-GK-DTPH prepared in Example 20 was diluted with deionized water to a concentration of 14 mg/ml In sterile syringe with a volume of 20 ml placed 11 ml CM-GK-DTPH. A solution of CM-GK-DTPH filtered through a sterile syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml. Prepare a solution of 50 mg/ml PEG-(acrylate)4[company Laysan Bio Inc., molecular weight 10000, the number 4arm-PEG-ACRL10K-1g] by dissolving 40,1 mg PEG-(acrylate)4in 0,802 ml of deionized water. A solution of PEG-(acrylate)4transferred into a sterile syringe with a volume of 1 ml and filtered through a sterile syringe filter with a pore size of 0.2 μm. Transfer 10 ml of sterile filtered CM-HADTPH in a sterile centrifuge tube with a volume of 50 ml In a solution of CM-GK-DTPH add 250 ál of a 0.5 M solution of sodium phosphate. The resulting solution was thoroughly mixed. Added to a solution of CM-GK-DTPH 560 µl [28 mg PEG-(acrylate)4] sterile solution of 50 mg/ml PEG-(acrylate)4. The resulting solution was thoroughly mixed. A solution of CM-GK-DTPH/PEG-(acrylate)4then placed in a thermostat with a temperature of 37°C for at least 16 hours. At this stage, a solution of CM-GK-DTPH/PEG-(acrylate)4stitched with gel formation. Then the gel-like material extracted from the components is the same. The gel-like material can be transformed into a suspension of the gel in the same manner as described in Example 3 and 4. The gel can be prepared in the presence of triamcinolone acetonide using the techniques described in Examples 9, 10 and 11. Hyaluronic acid can be added to the composition of the gel using the method described in Example 8. Hyaluronic acid can be added to the composition of the gel containing triamcinolone acetonide, using methods described in Example 13.

EXAMPLE 26

The SYNTHESIS of COMPOUNDS of HYALURONIC ACID-DITHIOBIS(DEHYDRATED PROPANOIC ACID) (GK-DTPH)

3,3'-Dithiobis(dehydrated propanoic acid) (DTPG) was synthesized according to previously published methods (Vercruysse, K. P.; Marecak, D. M.; Marecek, J. F.; Prestwich, G. D. "Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels of hyaluronic acid", Bioconjugate Chem. (1997) 8:686-694; Shu, X. Z.; Liu, Y.; Luo, Y.; Roberts, M. C; Prestwich, G. D. "Disulfide crosslinked hyaluronan hydrogels", Biomacromolecules(2002) 3: 1304-1311). 3,3'-Dithiobis(dehydrated propanoic acid) (16.7 g, 0.07 mol) was added to prepared above solution of hyaluronic acid (20 g of hyaluronic acid [Mwof 0.8-1.0 million] dissolved in 1000 ml deionized water), and adjusting the pH value of the solution to 4.75 by adding either HCl or NaOH solution. Then, added 0,384 g of 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC) [company Sigma-Aidrich], and supported the pH value of RA is down at a pH of 4.75 by addition of 6.0 N HCl under continuous stirring with a magnetic stirrer at room temperature. After 4 hours, was added 50 g of dithiothreitol (DTT) [company Biovectra], and adjusting the pH value of the solution to 8.5 by the addition of concentrated NaOH solution. Then after 12-24 hours of stirring with a magnetic stirrer at room temperature, adjusting the pH value of the reaction mixture to pH 3.0 by the addition of 6.0 N HCl. The acidified solution was purified and concentrated using flow filtering along the flow (TFF) using 20 l of 1 mm HCl, pH 3.0. The solution is then concentrated to a volume of approximately 1 liter

EXAMPLE 27

The SYNTHESIS GEL GK-DTPH/PEG-(ACRYLATE)2

The solution GK-DTPH prepared in example 26, diluted with deionized water to a concentration of 14 mg/ml In sterile syringe with a volume of 20 ml placed 11 ml solution GC-DTPH. The solution GK-DTPH filtered through a sterile syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml. Prepare a solution of 50 mg/ml PEG-(acrylate)2[company Laysan Bio Inc., molecular weight 3400, the catalog number of an ACRL-PEG-an ACRL-3400-1g] by dissolving 40,1 mg PEG-(acrylate)2in 0,802 ml of deionized water. A solution of PEG-(acrylate)2transferred into a sterile syringe with a volume of 1 ml and filtered through a sterile syringe filter with a pore size of 0.2 μm. Transfer 10 ml of sterile filtered solution GK-DPG in a sterile centrifuge tube with a volume of 50 ml To RA is Toru GK-DTPH add 250 ál of a 0.5 M solution of sodium phosphate. The resulting solution was thoroughly mixed. To a solution of GK-DTPH add 380 µl [19 mg PEG-(acrylate)2] sterile 50 mg/ml PEG-(acrylate)2. The resulting solution was thoroughly mixed. The solution GK-DTPH/PEG-(acrylate)2then placed in a thermostat with a temperature of 37°C for at least 16 hours. At this stage, the solution GK-DTPH/PEG-(acrylate)2stitched with gel formation. Then the gel-like material extracted from thermostat.

EXAMPLE 28

SUSPENSION GEL GK-DTPH/PEG-(ACRYLATE)2

Gel GK-DTPH/PEG-(acrylate)2(obtained in Example 27) is transformed into a suspension of the gel using the techniques described in Examples 3 and 4, respectively.

EXAMPLE 29

GEL GK-DTPH/PEG-(ACRYLATE)2,CONTAINING ACETONIDE TRIAMCINOLONE

A solution of CM-GK-DTPH prepared in Example 20 was diluted with deionized water to a concentration of 17.5 mg/ml was Placed 30 ml CM-GK-DPG in sterile syringe with a volume of 60 ml of a Solution of CM-GK-DTPH filtered through a sterile syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml was Added 100 mg of powder sterile triamcinolone acetonide, and the resulting mixture was thoroughly stirred. Prepare a solution of 40 mg/ml PEG-(acrylate)2[company Laysan Bio Inc., molecular weight 3400, the catalog number of an ACRL-PEG-ACRL3400-1g] by dissolving 600 mg of the EG-(acrylate) 2in 15 ml of a buffer of 0.2 M solution of sodium phosphate (pH 7,4). A solution of PEG-(acrylate)2transferred into a sterile syringe with a volume of 20 ml and filtered through a sterile syringe filter with a pore size of 0.2 μm. Transfer 20 ml of sterile filtered solution of CM-HADTPH in a sterile centrifuge tube with a volume of 50 ml To a solution of CM-GK-DTPH add 10 ml solution of PEG-(acrylate)2. The resulting solution was thoroughly mixed. Then the solution of CM-GK-DTPH/PEG-(acrylate)2give the ability to form a gel at room temperature.

The synthesis gel is repeated, using 33 mg, 50 mg, 75 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 375 mg, 400 mg and 500 mg of triamcinolone acetonide, respectively.

The gel is transformed into a suspension of the gel using the techniques described in Examples 3 and 4.

EXAMPLE 30

The SYNTHESIS GEL GK-DTPH/PEG-(ACRYLATE)4

The solution GK-DTPH obtained in Example 20 was diluted with deionized water to a concentration of 14 mg/ml In sterile syringe with a volume of 20 ml placed 11 ml solution GC-DTPH. The solution GK-DTPH filtered through a sterile syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 50 ml. Prepare a solution of 50 mg/ml PEG-(acrylate)4[company Laysan Bio Inc., molecular weight 10000, the number 4arm-PEG-an ACRL-10K-1g] by dissolving 40,1 mg PEG-(acrylate)4in 0,802 ml of deionized water. A solution of PEG(and is relat) 4transferred into a sterile syringe with a volume of 1 ml and filtered through a sterile syringe filter with a pore size of 0.2 μm. Transfer 10 ml of sterile filtered solution GK-DPG in a sterile centrifuge tube with a volume of 50 ml are Added to a solution of GK-DTPH 250 ál of a 0.5 M solution of sodium phosphate. The resulting solution was thoroughly mixed. To a solution of GK-DTPH add 560 µl [28 mg PEG-(acrylate)4] sterile solution of 50 mg/ml PEG-(acrylate)4. The resulting solution was thoroughly mixed. The solution GK-DTPH/PEG-(acrylate)4then placed in a thermostat with a temperature of 37°C for at least 16 hours. At this stage, the solution GK-DTPH/PEG-(acrylate)4stitched with gel formation. Then the gel-like material extracted from thermostat. The gel-like material can be transformed into a suspension of the gel in the same manner as described in Example 3 and 4. The gel can be prepared in the presence of triamcinolone acetonide using the techniques described in Examples 9, 10 and 11. Hyaluronic acid can be added to the composition of the gel using the method described in Example 8. Hyaluronic acid can be added to the composition of the gel containing triamcinolone acetonide, using methods described in Example 13.

EXAMPLE 31

PREPARATION of GEL PEG-DIACRYLATE (TESTED MATERIAL 4)

Download 1,466 g polietilen icol-diacrylate [PEG-DA] (Laysan Bio, the catalog number of an ACRL-PEG-an ACRL-3400-1g) in a sterile container with a volume of 125 ml For PEG-DA was added 22 ml of sterile physiological solution. After dissolution, filtered PEG-DA/NaCl through a syringe filter with a pore size of 0.2 μm disinfected in the Erlenmeyer flask. Filtered 0,150 M carbonate buffer with a pH of 8.2 through a syringe filter with a pore size of 0.2 μm, and 1 ml of this sterile solution was added to a solution of PEG-DA. The flask was closed with a rubber diaphragm, and from the solution was removed gases by barbotirovaniya through him nitrogen for 10 minutes. To the gas line was connected filter with a pore size of 0.2 μm to ensure the sterility of the air. Prepare a solution of 400 mg/ml of sodium ascorbate by adding 1.2 g of sodium ascorbate in a test tube of heat-resistant glass with a membrane cover. In a test tube was added 3 ml of deionized water. After dissolution, the solution was filtered through a syringe filter with a pore size of 0.2 μm in sterile centrifuge tube with a volume of 15 ml To a solution of PEG-DA was added 0.8 ml of sterile filtered solution of 400 mg/ml of sodium ascorbate. To a solution of PEG-DA was added to 0.8 ml of sterile filtered solution of 400 mg/ml sodium persulfate. The solution was stirred by education turbulence of the solution. The flask was closed with a red rubber membrane and the solution was removed gases by barbotin the cation through a solution of nitrogen for 15 minutes. To the gas line for supplying nitrogen, used for degassing of the solution was plugged filter with a pore size of 0.2 μm. The solution was placed in a thermostat with a temperature of 37°C for at least 18 hours for gel formation. The gel was transferred into a syringe with a volume of 30 ml was Cut from a sheet of mesh circular disk with a diameter of 23 mm with a punch to the skin with a diameter of 23 mm Disk from the grid was placed in a syringe filter made of polycarbonate with a diameter of 25 mm, which were removed by filtration layers. The gel was extrudible over the net (with holes of 1 mm×1 mm) in a glass of 250 ml To 25 ml extruded gel was added 100 ml of sterile physiological solution. After 40 minutes, the supernatant saline solution was decanted and added another 125 ml of sterile physiological solution. This procedure was repeated 3 times. After the last washing was added 45 ml of swollen gel to 45 ml of physiological solution, and the suspension is gently stirred. Adjust the pH of the obtained solution in the range of 7.0 to 7.4 using a combination of 1N NaOH and 1N HCl. 1.5 ml of this suspension gel was placed in a glass syringe with a volume of 5 ml was placed on the syringe cap. Then the syringe is sterilized in an autoclave at 250°C for 15 minutes.

EXAMPLE 32

PREPARATION of GEL PEG-DIACRYLATE/BESTIAL (test MATERIAL 2)

Downloaded 630 mg polyethylene glycol-diacrylate [PEG-DA] (firms who Laysan Bio, the catalog number of an ACRL-PEG-an ACRL-3400-1g) in a scintillation vial with a volume of 20 ml was Added 6 ml of deionized water. After dissolution, the solution was filtered through a syringe filter with a pore size of 0.2 μm. Dissolved 48 mg of N,N'-bis(acryloyl)applied (company Sigma, A4929) in 6 ml of tetrahydrofuran (THF) in a glass scintillation vial. After dissolution, the solution was mixed with a solution of PEG-DA. The vial was closed with screw-on membrane cap, and from the solution was removed gases by ozonation of nitrogen for 10 minutes. To a solution of PEG-DA was added 50 μl of a solution of 400 mg/ml of sodium ascorbate (prepared using deionized water and filtered through a syringe filter with a pore size of 0.2 μm). To a solution of PEG-DA was added 50 μl of a solution of 400 mg/ml sodium persulfate (prepared using deionized water and filtered through a syringe filter with a pore size of 0.2 μm). Return to the place of the membrane cover, and from the solution was removed gases by ozonation of nitrogen for 10 minutes. The solution was placed in a drying Cabinet, which was set temperature of 60°C. the Solution turned into a gel after 15 minutes. The gel was removed from the oven and cooled to room temperature. The gel was transferred into a syringe with a volume of 30 ml was Cut from a sheet of mesh circular disk with a diameter of 23 mm with a punch to the diameter 23 mm The drive from the grid was placed in a syringe filter made of polycarbonate with a diameter of 25 mm, which were removed by filtration layers. The gel was extrudible over the net (with holes ~0.8 mm×~0.8 mm) in a beaker with a volume of 400 ml To the extruded gel was added 200 ml of deionized water. After 45 minutes, the supernatant was poured and was added 200 ml of deionized water. This procedure was repeated 4 times. Then the stage of washing was repeated 3 times using a 0.9% saline solution. The supernatant was removed and the remaining gel was again extrudible through a grid (described above). 1.5 ml of this suspension gel was placed in a glass syringe with a volume of 5 ml was placed on the syringe cap. Then the syringe is sterilized in an autoclave at 250°C for 15 minutes.

EXAMPLE 33

PREPARATION of GEL PEG-(LYS)4(The test MATERIAL 3)

In a glass container with a volume of 60 ml was loaded with 1.0 g of PEG-(lys)4[4-tier polyethylene glycol (Mw10000), terminal hydroxyl groups of which have functionalliteracy using glutaric anhydride and then with lysine]. Added to PEG-(lys)434 ml of dichloromethane. To the solution was added 333 μl of diisopropylcarbodiimide (firm Ruka, 38370). The solution turned into a gel after about 30 minutes. Gave the opportunity to continue the process of gelation for at least 18 hours. Gel shift and into the syringe a volume of 30 ml. Cut from a sheet of mesh circular disk with a diameter of 23 mm with a punch to the skin with a diameter of 23 mm Disk from the grid was placed in a syringe filter made of polycarbonate with a diameter of 25 mm, which were removed by filtration layers. The gel was extrudible over the net (with holes ~0.38 mm×~0.38 mm) in a beaker with a volume of 400 ml was Washed with 33 ml of gel with 330 ml of acetone. After 30 minutes, the acetone was removed. The washing procedure was repeated 4 times. Then the gel was dried under vacuum (Approximately 18 hours under vacuum). Added 771 mg of the dried gel to 52 ml of physiological solution, and allowed the gel to swell for 5 hours. The gel is then punching through the mesh (with holes ~0.38 mm×~0.38 mm). 1.5 ml of this suspension gel was placed in a glass syringe with a volume of 5 ml was placed on the syringe cap. Then the syringe is sterilized in an autoclave at 250°C for 15 minutes.

EXAMPLE 34

IN-VIVO STUDY: intra-ARTICULAR INJECTION of CROSSLINKED ha-VS-PEG-(SH)2HYDROGEL CONTAINING CORTICOSTEROID

Slightly cross-linked hydrogel, prepared by the reaction of hyaluronic acid, modified vinylsulfonate, with PEG-dithiol (ha-VS-PEG-(SH)2containing corticosteroid, triamcinolone acetonide was injected with in the intra-articular space of the knee joint goats. To assess the local and systemic effects of such injections were conducted morphologies the e research and histological examination of the synovial fluid. Details of the study are given below.

A. the tested materials

The test material 1.GK-VS-PEG-(SH)2(cross-linked hydrogel-based Ledger, containing no drug) were prepared as follows.

Solution SC-SU, prepared in Example 1 was diluted with deionized water to a concentration of 14 mg/ml In sterile syringe with a volume of 20 ml was placed 11 ml solution GC-SU. The solution GK-sun filtered through a sterile syringe filter with a pore size of 0.2 μm sterile syringe with a volume of 20 ml to Prepare a solution of 50 mg/ml PEG - (SH)2by dissolving 40,1 mg PEG-(SH)2in 0,802 ml of deionized water. Endured a solution of PEG-(SH)2in sterile syringe with a volume of 3 ml, and filtered through a syringe filter with a pore size of 0.2 μm. Transferred to 10 ml of sterile filtered solution SC-SU in a sterile centrifuge tube with a volume of 50 ml solution GC-SU was added 250 μl of a 0.5 M solution of sodium phosphate. The resulting solution was thoroughly mixed. To a solution of SC-SU was added to 380 ml sterile solution (50 mg/ml PEG-(SH)2. The resulting solution was thoroughly mixed. Then a solution of ha-VS/PEG-(SH)2was placed in a thermostat with a temperature of 37°C for at least 16 hours. At this stage, a solution of ha-VS/PEG-(SH)2sewn together with the formation of the gel. Then the gel-like material was removed from the baths is a stat.

The test material 2.GK-VS-PEG-(SH)2-triamcinolone acetonide ("CC-g-PEG - (SH)2-TA) were prepared as follows.

Mixed of 100.2 mg of triamcinolone acetonide (company Sicor, pharmacopeias purity, fine) with 2 ml of deionized water in a scintillation vial with a volume of 20 ml. After dispersion with ultrasound for 20 minutes, the material was sterilized in an autoclave at 250°C for 15 minutes. Placed 9 ml solution GC-SU, prepared in Example 1, with the concentration of 18.3 mg/ml in sterile syringe with a volume of 20 ml Solution GC-SU was filtered through a syringe filter with a pore size of 0.2 μm sterile syringe with a volume of 10 ml to Prepare a solution of 50 mg/ml PEG-(SH)2by dissolving 35 mg PEG-(SH)2in 0.7 ml of deionized water. A solution of PEG(SH)2was transferred into a sterile syringe with a volume of 3 ml and filtered through a sterile syringe filter with a pore size of 0.2 μm. Transferred to 7.6 ml of sterile filtered solution SC-SU in the solution of triamcinolone acetonide. Was added into the vial containing the ha-sun and triamcinolone acetonide, 370 μl of deionized water and 250 µl of 0.5 M solution of sodium phosphate. The resulting solution was thoroughly mixed. To a solution of SC-SU/triamcinolone acetonide was added 380 ál sterile solution of 50 mg/ml PEG-(SH)2. The resulting solution was thoroughly mixed. Then the races is the thief Ledger-SU/acetonide triamcinolone/PEG-(SH) 2was placed in a thermostat with a temperature of 37°C for at least 16 hours. At this stage, a solution of ha-VS-PEG-(SH)2TA was sutured to the formation of the gel. Then the gel-like material was removed from thermostat.

The test material 3.Acetonide triamcinolone 2 mg/ml (medication Kenalog-10; 10 mg/ml of triamcinolone acetonide, diluted with saline to 2 mg/ml).

The test material 4.Acetonide triamcinolone, 8 mg/ml (medication Kenalog-40; 40 mg/ml triamcinolone acetonide, diluted with saline to 8 mg/ml).

Control.Saline solution, 0.9% sodium chloride.

All test materials before use and kept at room temperature. From each of the test or control material to prepare a dose of 1.5 ml for each individual intra-articular injection.

B. Animals

For this study used a total of 24 goats with a Mature skeleton. They were purchased from a source approved by the Ministry of agriculture of the United States. In the beginning of the study, the animals weighed from 63 to 97 pounds.

Goats were purchased from approved by the Ministry of agriculture U.S. source, and they were checked for the absence of goat arthritis-encephalitis (CAE) and diseases of John, before you expose them to study. For each animal was conducted examined the e General health (including visual inspection of the limbs, nastrugannoy respiration, and absence of diarrhoea and nasal discharge) by a qualified veterinarian before them to do research. Animals were examined for any sign of disease or claudication. The selection of animals in the group for the study was determined by the absence of disease, clinical health, and lack of use of the knee joint research in the past. Goats were subjected to acclimatization during the relevant period of time defined by the relevant standards. The conditions of the animal consistent with applicable laws and regulations related to laboratory animals. After injection of goats kept inside the premises of large pastures (paddocks). During the whole time the goats were in conditions of free-riding behavior.

All animals received approximately 2 pounds a day diet for a small some animal, and unground hay. Access to piped water was unlimited. Feeding was stopped for approximately 12-24 hours before anesthesia, and access to water was stopped for approximately 12 hours prior to injection.

Animals were daily examined on the subject of overall health throughout the course of the study. If the animals showed any signs of postoperative complications or other signs of illness evania, pain or stress, were taken appropriate measures. In addition, in the unlikely event of injury, illness, or agony of the animal, provided medical care in accordance with current veterinary practice.

C. Therapy

The study was planned as follows.

Table 8
Groups and assigned therapy
GroupEar tagRight knee
(1.5 ml)
Left knee
(1.5 ml)
The time of killing the animal after injection into the right knee injection in his left knee
13171The test material 1Saline14 days
13256The test material 1Saline14 days
13831The test material 1Saline14 days
23174The test material 2Saline14 days
23596The test material 2Saline14 days
23840The test material 2Saline14 days
33133The test material 3Saline14 days
33177The test material 3Saline14 days
33833The test material 3Saline14 days
43589The test material 4Physiological age is the PR 14 days
43593The test material 4Saline14 days
43849The test material 4Saline14 days
53267The test material 1Saline28 days
53595The test material 1Saline28 days
53837*The test material 1Saline28 days (24 days*)
63173The test material 2Saline28 days
63264 The test material 2Saline28 days
63587The test material 2Saline28 days
73591The test material 3Saline28 days
73592The test material 3Saline28 days
73594The test material 3Saline28 days
83162The test material 4Saline28 days
83588*The test material 4Saline28 days (19 days*)
83590The test material 4Saline28 days
*animal died prematurely; (total number of days of participation in the study).

Each animal was measured body mass, size, joint circumference, and the amplitude of motion of the joint before injection (Day 1) and immediately before sacrifice (Day 14 or 28).

The main method of injection was the same for all subjects. All injections were performed under strict sterile. Animals were anestesiologi by intravenous injection of diazepam (0.1-0.5 mg/kg) and ketamine (4,4-7.5 mg/kg). Every knee was physically examined for the knockout, range of motion, swelling, temperature, crepitation, on the trajectory of the patella, and valgus/varus violations.

All injections were performed using conventional aseptic techniques. The left and right knee were prepared for injection by vystrogana wool in their respective fields and then disinfect them by careful treatment with chlorhexidine. The animal was transferred to the supine position. Right knee was dezinficiruy three times by careful treatment with chlorhexidine, carebusiness treatment with 70% isopropyl alcohol, and stained with iodine solution.

For injections in each knee joint used a standardized methodology. A sterile needle 21 size length of 2 inches was introduced in the intra-articular space through the anteromedial access. Injected the needle until it touches the side wall mimimally cutting the medial femoral condyle and gently took her back. Were injected with the right joint, 1.5 ml of the appropriate test material. The injection needle was removed, and was supported by increased pressure in the injection. Injected knee then 20 times were subjected to a full cycle on "bending". Immediately after this, the left knee disinfected three times by careful treatment with chlorhexidine, alternating treatment with 70% isopropyl alcohol and stained with iodine solution, and were injected with left knee 1.5 ml Control material in the same manner as described above for the right knee. The injection needle was removed, and was supported by increased pressure in the injection. Injected knee then 20 times were subjected to a full cycle on "bending".

After the injection was tested those animals that showed signs of distress and discomfort, and if necessary, they have introduced additional analgesic. All metadiorite recorded in the relevant documentation on the study.

D. Analysis

Blood samples are taken:Took blood from each animal immediately before the beginning of the study, and 5 hours after injection and after 1, 4, 7, 14 and 28 days for each of the remaining animals. At any given time clinical analysis of blood and chemical analysis of the blood.

Necropsy:Animals were killed humanely or 14 days, or 28 days after the start of injection by intravenous diazepam 0.22 mg/kg and ketamine 10 mg/kg to achieve a state of General anesthesia. Then shot the animals were injected intragastrically lethal dose of concentrated solution of potassium chloride (KCl) as long as not stated cardiac arrest.

Visual morphological observations:After collecting the knee joints were opened and conducted are described in table 9 visual inspection injected knee joints.

Table 9
Visual assessment and collection of samples
SampleVisual assessmentCollecting samplesPhotography and points
Synovial fluid (left and right)XX
Left and right knee jointsX
Left and right rear synovial bagXXX
A sample of the left and right cartilageXXX
Left and right popliteal lymph nodesXXX

In addition, conducted a semi-quantitative estimation of the joint with a single expert, as shown in Table 10.

Table 10
Qualitative assessment based on visual inspection
PointsStainingHyperemiaSwelling
0NormalNoNo
1Slightly yellowehka Light
2YellowModerateModerate
3VisibleVisible

The total number of points in the visual inspection was the sum of the scores of staining, hyperemia, and edema (0-8 points).

Evaluation of synovial fluid

After collecting synovial fluid from open joints, which recorded the total volume. Visual and semiquantitative scoring was assessed viscosity, transparency and the color of the liquid, as shown in Table 11. Using hemocytometer determined the total number of leukocytes. In addition, a smear was prepared synovial fluid for the differential microscopic analysis. The remaining synovial fluid was kept frozen in cryovials with individual labels at -80°C. Smear synovial fluid was retained for possible future analysis.

Table 11
Description and scoring of synovial fluid
PointsColor TransparencyTendon
0S=STRAWC=TRANSPARENTN=NORMAL
1P=PINKH=WHITEA=ABNORMAL
2Y=YELLOW R=REDD=CLOUDYW=WATERY
3B=BLOODYT=OPAQUE

The total number of points for synovial fluid was the sum of the scores of color, transparency and tendons (0-8 points).

Histological evaluation:

Immediately after dissection and subsequent visual evaluation of the surface of the joint was made sagittal slice of the medial femoral condyle (MFC) of each joint. Each of these sections were placed in 10% neutral buffered formalin. Fixed tissue was sent using overnight delivery to the company Premier Laboratories for processing. Sections of the right and left medial femoral condyle was subjected processed using standard histological techniques and stained with hematoxylin who eosin (H& E) and safranin-O (SAF-O) control staining dye green solid. Microscopic preparations slices of the medial femoral condyle was assessed using the assessment system Mancina in points for osteoarthritis in table 12.

Table 12
Modified assessment system Mancina in points
StructureNormal [0]
Surface roughness [1]
The pannus and surface irregularities [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
CellNormal [0]
The high content of diffuse parenchymal cells [1]
Cloning [2]
Deficiency of cells in the tissue [3]
The safranin-ONormal [0]
A small decrease in staining [1]
A moderate decrease in staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Assessment of the integrityDamage is absent [0]
Crossing blood vessels [1]
The maximum number of points14 (Rate=0)

E. Results

The results of the evaluation points Mancina for samples of cartilage of joints subjected to treatment with various compositions shown in tables 13a-h and are shown in Figure 11. In Figure 9 (14 days after therapy) and Figure 10 (28 after therapy) the scoring stained with safranin-O samples of cartilage of joints subjected to treatment using the compositions, after 14 and 28 days after the treatment, respectively.

Table 13a
Scoring femoral cartilage system Mancina 14 days, group 1
SampleTherapyStructureCellThe safranin-OAssessment of the integrityThe total number of points on the evaluation Mancina
3256
RFC
Gel SC-SU532010
3171
RFC
Gel SC-SU41005
3831
RFC
Gel SC-SU00101
AVERAGE3,01,31,00,0The STANDARD DEVIATION2,61,51,00,04,5
3256
LFC
0.9% NaCl41207
3171
LFC
0.9% NaCl41005
3831
LFC
0.9% NaCl00101
AVERAGE2,70,71,00,04,3
The STANDARD DEVIATION2,30,61,00,04,1

MODIFIED ASSESSMENT SYSTEM MANCINA IN POINTS

STRUCTURECELLThe SAFRANIN-OAssessment of the INTEGRITY
Normal [0]
Bumps
surface [1]
More widespread surface roughness [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
Normal [0]
Srednevekovyi and multifocal deficits cells [1]
Srednevekovyi and multifocal deficits cells cloning [2]
Intense focal and diffuse deficits cells [3]
Normal [0]
A small decrease in staining [1]
A moderate decrease in staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Damage is absent [0]
Crossing blood vessels [1]

Table 13b
Scoring femoral cartilage system Mancina 14 days, group 2
SampleTherapy StructureCellThe safranin-OAssessment of the integrityThe total number of points on the evaluation Mancina
3840
RFC
Gel SC-SU/TA3,01,02,00,06,0
3596
RFC
Gel SC-SU/TA0,00,03,00,03,0
3174
RFC
Gel SC-SU/TA4,02,02,00,08,0
AVERAGE2,31,02,30,0the 5.7
The STANDARD DEVIATION2,11,00,6 2,5
3840
LFC
0.9% NaCl3,01,00,00,04,0
3596
LFC
0.9% NaCl0,00,01,00,01,0
3174
LFC
0.9% NaCl4,02,02,00,08,0
AVERAGE2,31,01,00,04,3
The STANDARD DEVIATION2,11,01,00,03,5

MODIFIED ASSESSMENT SYSTEM MANCINA IN POINTS

STRUCTURECELL The SAFRANIN-OAssessment of the INTEGRITY
Normal [0]
Surface roughness [1]
More widespread surface roughness [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
Normal [0]
Srednevekovyi and multifocal deficits cells [1]
Srednevekovyi and multifocal deficits cells cloning [2]
Intense focal and diffuse deficits cells [3]
Normal [0]
A small decrease in staining [1]
A moderate decrease in staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Damage is absent [0]
Crossing blood vessels [1]

Table 13c
Scoring femoral cartilage system Mancina 14 days, group 3
SampleTherapyStructureCellThe safranin-OAssessment of the integrityThe total number of points in si is the subject of the evaluation Mancina
3833
RFC
Triamcinolone
acetonide
2 mg/ml
4,01,03,00,08,0
3133
RFC
Triamcinolone
acetonide
2 mg/ml
0,00,04,00,04,0
3177
RFC
Triamcinolone
acetonide
2 mg/ml
4,01,02,00,07,0
AVERAGE2,70,73,00,06,3
The STANDARD DEVIATION2,30,61,00,02,1
3833
LFC
0.9% NaCl0,0 0,01,00,01,0
3133
LFC
0.9% NaCl0,00,02,00,02,0
3177
LFC
0.9% NaCl0,00,01,30,01,0
AVERAGE0,00,01,30,01,3
The STANDARD DEVIATION0,00,00,60,00,6

MODIFIED ASSESSMENT SYSTEM MANCINA IN POINTS

STRUCTURECELLThe SAFRANIN-OAssessment of the INTEGRITY
Normal [0]
Roughness is poverhnosti [1]
More widespread surface roughness [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
Normal [0]
Srednevekovyi and multifocal deficits cells [1]
Srednevekovyi and multifocal deficits cells cloning [2]
Intense focal and diffuse deficits cells [3]
Normal [0]
A small decrease in staining [1]
A moderate decrease in staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Damage is absent [0]
Crossing blood vessels [1]

Table 13d
Scoring femoral cartilage system Mancina 14 days, group 4
SampleTherapyStructureCellThe safranin-OAssessment of the integrityThe total number of points on the evaluation Mancina
3593
RFC
Triamcinolone
acetonide
8 mg/ml
0,0 0,04,00,04,0
3849
RFC
Triamcinolone
acetonide
8 mg/ml
5,01,03,00,09,0
3589
RFC
Triamcinolone
acetonide
8 mg/ml
0,00,03,00,03,0
AVERAGE1,70,33,30,05,3
The STANDARD DEVIATION2,90,60,60,03,2
3593
LFC
0.9% NaCl0,00,02,00,02,0
3849
LFC
0.9% NaCl1,01,02,00,04,0
3589
LFC
0.9% NaCl0,00,03,00,03,0
AVERAGE0,30,32,30,03,0
The STANDARD DEVIATION0,60,60,60,01,0

MODIFIED ASSESSMENT SYSTEM MANCINA IN POINTS

STRUCTURECELLThe SAFRANIN-OAssessment of the INTEGRITY
Normal [0]
Surface roughness [1]
More widespread surface roughness [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
Normal [0]
Srednevekovyi and multifocal deficits cells [1]
Srednevekovyi and multifocal deficits cells cloning [2]
Intense focal and diffuse deficits cells [3]
Normal [0]
A small decrease in staining [1]
A moderate decrease in staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Damage is absent [0]
Crossing blood vessels [1]

Table 13e
Scoring femoral cartilage system Mancina after 28 days; group 5
SampleTherapyStructureCellThe safranin-OAssessment of the integrityThe total number of points on the evaluation Mancina
3267
RFC
Gel
GK-SU
11103
3837
RFC*
Gel
GK-SU
31307
3595
RFC
Gel
GK-SU
11103
AVERAGE1,01,01,00,03,0
The STANDARD DEVIATION0,00,00,00,00,0
3267
LFC
0.9% NaCl11103
3837
LFC*
0.9% NaCl31307
3595
LFC
of 0.9% NaC 11204
AVERAGE1,01,01,50,03,5
The STANDARD DEVIATION0,00,00,70,00,7
*Animal died prematurely. Data registered but not included in the calculation of the average value

MODIFIED ASSESSMENT SYSTEM MANCINA IN POINTS

STRUCTURECELLThe SAFRANIN-OAssessment of the INTEGRITY
Normal [0]
Surface roughness [1]
More widespread surface roughness [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
Normal [0]
With epochally and multifocal deficits cells [1]
Srednevekovyi and multifocal deficits cells cloning [2]
Intense focal and diffuse deficits cells [3]
Normal [0]
A small decrease in staining [1]
A moderate decrease in staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Damage is absent [0]
Crossing blood vessels [1]

0,0
Table 13f
Scoring femoral cartilage system Mancina after 28 days, the group 6
SampleTherapyStructureCellThe safranin-OAssessment of the integrityThe total number of points on the evaluation Mancina
3264
RFC
Gel
GK-SU/TA
0,00,03,00,03,0
3587
RFC
Gel
GK-SU/TA
5,02,02,09,0
3173
RFC
Gel
GK-SU/TA
0,00,02,00,02,0
AVERAGE1,70,72,30,0the 4.7
The STANDARD DEVIATION2,91,20,60,0the 3.8
3264
LFC
0.9% NaCl0,00,00,00,00,0
3587
LFC
0.9% NaCl5,02,01,00,08,0
3173
LFC
0.9% NaCl0,00,01,0 0,01,0
AVERAGE1,70,70,70,03,0
The STANDARD DEVIATION2,91,20,60,04,4

MODIFIED ASSESSMENT SYSTEM MANCINA IN POINTS

STRUCTURECELLThe SAFRANIN-OAssessment of the INTEGRITY
Normal [0]
Surface roughness [1]
More widespread surface roughness [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
Normal [0]
Srednevekovyi and multifocal deficits cells [1]
Srednevekovyi and multifocal deficits cells cloning [2]
Intense focal and diffuse deficits cells [3]
Normal [0]
A small decrease in staining [1]
At erenee reduced staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Damage is absent [0]
Crossing blood vessels [1]

Table 13g
Scoring femoral cartilage system Mancina after 28 days, the group 7
SampleTherapyStructureCellThe safranin-OAssessment of the integrityThe total number of points
the evaluation system Mancina
3592
RFC
Triamcinolone
acetonide
2 mg/ml
4,03,03,00,010,0
3591
RFC
Triamcinolone
acetonide
2 mg/ml
0,02,04,00,06,0
3594
RFC
Triamcinolone
acetonide
2 mg/ml
4,0 2,03,00,09,0
AVERAGE2,72,33,30,08,3
The STANDARD DEVIATION2,30,60,60,02,1
3592
LFC
0.9% NaCl4,01,01,00,06,0
3591
LFC
0.9% NaCl1,01,01,00,03,0
3584
LFC
0.9% NaCl2,01,01,00,04,0
AVERAGE2,31,0 1,00,04,3
The STANDARD DEVIATION1,50,00,00,01,5

MODIFIED ASSESSMENT SYSTEM MANCINA IN POINTS

STRUCTURECELLThe SAFRANIN-OAssessment of the INTEGRITY
Normal [0]
Surface roughness [1]
More widespread surface roughness [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
Normal [0]
Srednevekovyi and multifocal deficits cells [1]
Srednevekovyi and multifocal deficits cells cloning [2]
Intense focal and diffuse deficits cells [3]
Normal [0]
A small decrease in staining [1]
A moderate decrease in staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Damage is absent [0]
The Peres is a group of blood vessels [1]

Table 13h
Scoring femoral cartilage system Mancina after 28 days, the group 8
SampleTherapyStructureCellThe safranin-OAssessment of the integrityThe total number of points on the evaluation Mancina
3588
RFC*
Trianti-Nolan
acetonide
8 mg/ml
4,03,03,00,010,0
3590
RFC
Trianti-Nolan
acetonide
8 mg/ml
5,03,04,00,0to 12.0
3162
RFC
Trianti-Nolan
acetonide
8 mg/ml
1,01,04,00,06,0
AVERAGE 3,02,04,00,09,0
The STANDARD DEVIATION2,81,40,00,04,2
3588
LFC*
0.9% NaCl4,03,03,00,010,0
3590
LFC
0.9% NaCl3,01,02,00,06,0
3162
LFC
0.9% NaCl3,01,02,00,06,0
AVERAGE3,01,02,00,06,0
The STANDARD DEVIATION 0,00,00,00,00,0
*Animal died prematurely. Data registered but not included in the calculation of the average value

MODIFIED ASSESSMENT SYSTEM MANCINA IN POINTS

STRUCTURECELLThe SAFRANIN-OAssessment of the INTEGRITY
Normal [0]
Surface roughness [1]
More widespread surface roughness [2]
The cracks in the transition zone [3]
Cracks in the radial zone [4]
Cracks in soda zone [5]
Complete disorganization [6]
Normal [0]
Srednevekovyi and multifocal deficits cells [1]
Srednevekovyi and multifocal deficits cells cloning [2]
Intense focal and diffuse deficits cells [3]
Normal [0]
A small decrease in staining [1]
A moderate decrease in staining [2]
A strong decrease in staining [3]
Staining is absent [4]
Damage is absent [0]
Crossing the blood the blood vessels [1]

In the cartilage on the 28th day, there was no difference in the estimates for the modified system Mancina for gel ha-VS-PEG-(SH)2and control material. Observed loss in the intensity of staining with safranin-O, expressed a moderate increase in part in scores on the modified system Mancina for groups 7 and 8 compared with either group 5 or group 6. In the period from 14 days to 28 was not observed increase in scores on the modified system Mancina for both groups subjected to therapy only one triamcinolone acetonide (groups 7 and 8). This increase in scores over time was not observed for group 5, subjected to a treatment using gel ha-VS-PEG-(SH)2or for a group of 6, subjected to treatment using gel ha-VS-PEG-(SH)2-TA.

The results of this study indicate the absence of local and systemic effects after 28 days after intra-articular injection of 1.5 ml only gel ha-VS-PEG-(SH)2or gel ha-VS-PEG-(SH)2-TA, combined with 2 mg/ml of triamcinolone acetonide in the knee goats. Negative effects on cartilage included in the gel ha-VS-PEG-(SH)2the triamcinolone acetonide was smaller than the impact of the injection of an equivalent dose or a higher dose of only one of triamcinolone acetonide.

Specific staining of g is casemanagement using safranin-O showed what negative effect on the cartilage of triamcinolone acetonide at a concentration of 2 mg/ml (3 mg) in combination with gel ha-VS-PEG-(SH)2it was less than 2 mg/ml (3 mg) and 8 mg/ml (12 mg) bolus dose of triamcinolone acetonide on day 14 and 28 days after the injection.

In Fig. 12 and 13 shows a typical stained with safranin-O histological specimens (40X) of the medial femoral condyle after 14 days (Fig. 12) and 28 days (Fig. 13) after injection. The figures shows that there is a more intense safranin-O for sample cartilage of joints subjected to therapy with triamcinolone acetonide included in the hydrogel than for sample cartilage subjected to treatment using an equivalent dose of only one of triamcinolone acetonide, which were injected with directly into the joint (i.e., not included in the hydrogel).

EXAMPLE 35

FORCE MEASUREMENT EXTRUSION

The force required for extrusion of the product GK-VS/PEG-(SH)2/GK (Example 5, Example 41) and product GK-VS/PEG-(SH)2/GC with triamcinolone acetonide (Example 38), was measured using the installation with the actuator for measuring the strength of Chatillon (installation of electric Chatillon LTCM-6 digital instrument measuring the strength of Chatillon DFE-025, firms Ametec TCI Division). A fixture for fixing the syringe of 10 ml were mounted on a base plate installation with electronic what troprivoda thus, to the measured force was located directly over the piston rod of the syringe. Included installation and set the value of the speed of 3 inches per minute by setting the drive speed control to position "3". Moved console engine installation at the highest point. Remove the cap from the glass syringe of 10 ml, which contained exposed to the test composition, and would put the needle 21 size to an open end of the syringe. After the syringe was placed in a holder for a syringe, console engine installation slowly moved down until the sensor measuring the strength of slightly does not touch the stem of the piston of the syringe. Under the tip of the needle 21 size was put to a test tube with a volume of 16 ml Set the unit of measurement of force for the registration of the maximum force. Was zeroed the readings of the force measurement. Then pressed the toggle switch installation, resulting in pressure was exerted on the plunger of the syringe, and the contents of the syringe were being squeezed through a needle 21 size. The installation with the drive stopped immediately before the moment of achievement of bounding the bottom of the syringe. Recorded the maximum extrusion force displayed on the display screen of the device for measuring force. The results of tests of various compositions are given below:

Table 14
No. sampleThe force of extrusion (lbs)
GK-VS/PEG-(SH)2with the Ledger
(Series NB30:16)
GK-VS/PEG-(SH)2with the Ledger
(Series M0229)
GK-VS/PEG-(SH)2/TA and SC
(Series M0231)
16,910,52the 6.06
27,9of 10.217,78
36,110,368,14
47,2
56,9
67,3
AVERAGE7,110,367,33
The STANDARD DEVIATION 0,60,161,11

EXAMPLE 36

The CONSTANT FORCE of EXTRUSION GK-VS/PEG-(SH)2With Ledger IN TIME

The force required for extrusion of the product GK-VS/PEG-(SH)2/GK (Example 5), as a function of time was measured using the installation with the actuator for measuring the strength of Chatillon (installation of electric Chatillon LTCM-6 digital instrument measuring the strength of Chatillon DFE-025, firms Ametec TCI Division). Effect of extrusion was measured immediately after it was cooked product, and then after 1 month and 3 months after the initial measurement. The samples were stored at room temperature for 3 months. Effect of extrusion was measured for each sample as follows. A fixture for fixing the syringe of 10 ml were mounted on a base plate installation with the actuator so that the measured force was located directly over the piston rod of the syringe. Included installation and set the value of the speed of 3 inches per minute by setting the drive speed control to position "3". Moved console engine installation at the highest point. Remove the cap from the glass syringe of 10 ml, which contained exposed to the test composition, and would put the needle 21 size to an open end of the syringe. After the syringe was placed in a holder for a syringe,console engine installation slowly moved down until while the sensor of the instrument measuring the strength of slightly does not touch the stem of the piston of the syringe. Under the tip of the needle 21 size was put to a test tube with a volume of 16 ml Set the unit of measurement of force for the registration of the maximum force. Was zeroed the readings of the force measurement. Then pressed the toggle switch installation, resulting in pressure was exerted on the plunger of the syringe, and the contents of the syringe were being squeezed through a needle 21 size. The installation with the drive stopped immediately before the moment of achievement of bounding the bottom of the syringe. Recorded the maximum extrusion force displayed on the display screen of the device for measuring force. The results for series NB30:16 below, and they show that there is no real change in the force required for extrusion of the product through the needle 21 size for 3 months.

Table 15
No. sampleThe force of extrusion (lbs)
T=0T=1 monthT=3 months
110,316,879,46
28,7a 7.627,14
38,7210,5511,57
4for 9.477,53of 7.36
510,438,25the 7.65
69,638,38,95
AVERAGE9,548,198,69
The STANDARD DEVIATION0,751,271,69

EXAMPLE 37

PREPARATION of the GEL of ha-VS/PEG-(SH)2/TA and SC

Download 170,8 mg, 341,3 mg, 511,7 mg, and 682,7 mg of sterile triamcinolone acetonide in 4 separate sterile plastic containers with a volume of 125 ml, which was marked as TA 10 mg, TA 20 mg, TA 30 mg and TA 40 mg, respectively. Each container containing TA, weighed on the scales and in each of the 4 tanks were added 14,98 g, 14,99 g, 14,99 g and 15,01 g of sterile filtered (filtered through sterile f is ltry with pore size 0.2 μm, PVDF membrane) solution of 14 mg/ml ha-sun in the water in order from TA 10 mg to THE 40 mg Powder TA and the solution Ledger-SU were mixed by stirring up until you get the solutions did not look visually homogeneous. In each capacity was added to the 0.375 ml of sterile filtered through a sterile filter with a pore size of 0.2 μm) of 1M solution of sodium phosphate, pH 7.4 and the mixture was thoroughly stirred. In each capacity was added 0,543 ml 50 mg/ml PEG-dithiol 3350 [PEG - (SH)2] (sterile filtered through a sterile filter with a pore size of 0.2 μm) and thoroughly mixed. Above the stage was implemented in Boxing for conducting biological research. The mixture was placed overnight in an oven with a temperature of 37°C. the Composition was removed from the drying chamber, the outer part of the tank rubbed with a mixture of 70/30 isopropanol/water and then transferred into the pits to conduct biological research. Each gel was crushed with sterile sticks. In containers labeled as TA 10 mg, 20 mg, 30 mg and 40 mg, respectively, was added 86,23 g, 86,25 g, 86,40 g and 86,23 g of the solution 7,83 mg/ml ha in 0.9% saline solution (filtered through a sterile filter with a pore size of 0.2 MK, PVDF membrane). Each mixture was allowed to swell at room temperature for 3 hours. Each mixture is ATEM was passed through a mesh hole size of 0.85 μm, installed in the filter housing [Cut a disk from a polyester mesh with a diameter of 23 mm (company McMaster Carr, part number 9218T13, the hole dimensions: 20,3 x 20,3, the size of the squares/rectangles: 0,0331", the nominal value in microns: 840 microns, the proportion living section: 46, the diameter of the thread: 0,0157") with a punch to the skin with a diameter of 23 mm Disk was inserted into the holder syringe filter 25 mm diameter (Cole Palmer, catalog number EW-29550-42), and the filter holder was closed. The filter holder containing the grid, sterilized in an autoclave]. Collected after passing through the grid the mixture was then again passed through the mesh hole size of 0.85 μm. The collected mixture is then kept in a plastic container.

EXAMPLE 38

PACKING GEL GK-VS/PEG-(SH)2/TA and SC

Then 6 ml of each composition of Example 37 was transferred to a glass syringe with a volume of 10 ml glass syringe BD Hypak, P/N 47262119), which had a cap. The piston rod is put in the rear end of the sterile tube (BD P/N 47318319), after which the tube/piston inserted into the body of the syringe. The syringe is turned upside down and the cap of the syringe slightly weakened. He pushed the piston up until the syringe was not removed a large part of the air. Then on the syringe tightly wore a cap. Above the stage was implemented in Boxing for conducting biological research. The process was repeated until the PRS, until it was put up the whole product.

EXAMPLE 39

DETERMINATION of PARTICLE SIZE - RINSING with DEIONIZED WATER

Stainless steel sieve hole size 2.36-mm (control sieve #8 on the US standard), 1.4 mm (control sieve #14 on the US standard), 1 mm (control sieve #18 on the U.S. standard), 0.85 mm (control sieve #20 on the US standard), 0.6 mm (control sieve #30 U.S. standard), 0,425 mm (control sieve #40 U.S. standard), 0.25 mm (control sieve #60 U.S. standard), and 0.150 mm (control sieve #100 U.S. standard) was washed with deionized water, and wiped dry with a lint-free wipes Kimwipes. After determining the mass of each sieve were installed at each other, starting with the smallest size (#100) at the bottom to the very large size (#8) at the top. In the top sieve slowly poured into 100 ml of the composition GK-VS/PEG-(SH)2/TA and SC (Example 37). After a large part of the liquid component of the sample passed through the upper sieve, was slowly added to the top sieve of approximately 50 ml of deionized water in order to wash out the component of the gel, which was detained on the sieve. After the liquid component is passed through the sieve, the sieve was removed from the stack. This process is repeated as long as each sieve was not washed and removed from the stack. Drops of excess water that remained on each sieve were removed the use of toilet paper. Determined the total mass of each sieve (the weight of the sieve and the delayed gel). Expected mass of the gel particles detained on each sieve by subtracting the original weight of the sieve from the total mass of the sieve. Expected percentage of detained each sieve gel by dividing the mass of the gel, the detainee on a particular sieve, by total weight of the gel, the detainee on all screens.

20
Table 16
Size sieve #Size of sieve (mm)The gel retained on each sieve (%)
TA 10TA 20TA 30TA40
82,360,10,30,50,0
141,4011,414,719,47,6
181,0044,948,034,235,2
0,8529,423,122,028,0
300,607,47,814,714,7
400,4253,72,74,47,8
600,251,51,72,94,2
1000,151,71,72,02,6

EXAMPLE 40

DETERMINATION of PARTICLE SIZE - FLUSHING saline

Stainless steel sieve hole size 2.36-mm (control sieve #8 on the US standard), 1.4 mm (control sieve #14 on the US standard), 1 mm (control sieve #18 on the U.S. standard), 0.85 mm (control sieve #20 on the US standard), 0.6 mm (control sieve #30 U.S. standard), 0,425 mm (control sieve #40 U.S. standard), 0.25 mm (control sieve #60 standard is the United States), and 0.150 mm (control sieve #100 U.S. standard) was washed with deionized water, and wiped dry with a lint-free wipes Kimwipes. After determining the mass of each sieve were installed at each other, starting with the smallest size (#100) at the bottom to the very large size (#8) at the top. In the top sieve slowly poured into 100 ml of the composition GK-VS/PEG-(SH)2/TA and SC (TA 10) (Example 37). After a large part of the liquid component of the sample passed through the upper sieve, was slowly added to the top sieve of approximately 50 ml of 0.9% saline solution to wash out the component of the gel, which was detained on the sieve. After the liquid component is passed through the sieve, the sieve was removed from the stack. This process is repeated as long as each sieve was not washed and removed from the stack. Drop excess saline solution, which remained on each sieve was removed with toilet paper. Determined the total mass of each sieve (the weight of the sieve and the delayed gel). Expected mass of the gel particles detained on each sieve by subtracting the original weight of the sieve from the total mass of the sieve. Expected percentage of detained each sieve gel by dividing the mass of the gel, the detainee on a particular sieve, by total weight of the gel, the detainee on all screens.

Table 17
Size sieve #Size of sieve (mm)The gel retained on each sieve (%)
Experiment-1Experiment-2Experiment 3AVERAGEThe STANDARD DEVIATION
82,360,00,40,70,40,3
141,409,03,410,57,6the 3.8
181,0050,650,148,8to 49.90,9
200,8520,523,218,620,82,3
30/td> 0,6010,912,810,9the 11.61,1
400,4254,85,2the 5.75,30,4
600,252,73,53,13,10,4
1000,151,31,11,71,40,3

EXAMPLE 41

SUSPENSION GEL of ha-VS/PEG-(SH)2With HYALURONIC ACID - Ledger SWELLING

Each of the three sterile containers with a volume of 125 ml was weighed on the scales and was added in each of the three tanks 14,97 g, 14.95 for g and 15.00 g of a solution of 14 mg/ml ha-sun in the water (filtered through a sterile filter with a pore size of 0.2 µm PVDF membrane). In each capacity was added to the 0.375 ml of sterile filtered through a sterile filter with a pore size of 0.2 μm) of 1M solution of sodium phosphate, pH 7.4 and carefully what about mixed. In each capacity was added 0,543 ml 50 mg/ml PEG - (SH)2(sterile filtered through a sterile filter with a pore size of 0.2 μm) and thoroughly mixed. Above the stage was implemented in Boxing for conducting biological research. The mixture was placed overnight in an oven with a temperature of 37°C. the Composition was removed from the drying chamber, the outer part of the tank rubbed with a mixture of 70/30 isopropanol/water and then transferred into the pits to conduct biological research. Each gel was crushed with sterile sticks. Then the vessel was added 86,40 g, 86,21 g and 86,31 g of the solution 7,83 mg/ml ha in 0.9% saline solution (filtered through a sterile filter with a pore size of 0.2 MK, PVDF membrane). Gels swollen at room temperature for 3 hours. Each mixture was then passed through a mesh hole size of 0.85 μm, installed in the filter housing [Cut a disk from a polyester mesh with a diameter of 23 mm (company McMaster Carr, part number 9218T13, the hole dimensions: 20,3 x 20,3, the size of the squares/rectangles: 0,0331", the nominal value in microns: 840 microns, the proportion living section: 46, the diameter of the thread: 0,0157") with a punch to the skin with a diameter of 23 mm Disk was inserted into the holder syringe filter 25 mm diameter (Cole Palmer, catalog number EW-29550-42), and the filter holder was closed. erately filter, which contained the grid, sterilized in an autoclave]. Collected after passing through the grid the mixture was then again passed through the mesh hole size of 0.85 μm. The collected mixture is then kept in a plastic container.

EXAMPLE 42

PACKING SUSPENSION GEL of ha-VS/PEG-(SH)2With HYALURONIC ACID

Then 6 ml of each composition of Example 41 was transferred to a glass syringe with a volume of 10 ml glass syringe BD Hypak, P/N 47262119), which had a cap. The piston rod is put in the rear end of the sterile tube (BD P/N 47318319), after which the tube/piston inserted into the body of the syringe. The syringe is turned upside down and the cap of the syringe slightly weakened. He pushed the piston up until the syringe was not removed a large part of the air. Then on the syringe tightly wore a cap. Above the stage was implemented in Boxing for conducting biological research. The process was repeated until then, until it was put up the whole product.

EXAMPLE 43

DETERMINATION of PARTICLE SIZE - FLUSHING saline

Stainless steel sieve hole size 2.36-mm (control sieve #8 on the US standard), 1.4 mm (control sieve #14 on the US standard), 1 mm (control sieve #18 on the U.S. standard), 0.85 mm (control sieve #20 on the US standard), 0.6 mm (control sieve #30 U.S. standard), 0,425 mm (control sieve #40 U.S. standard), of 0.25 is m (control sieve #60 U.S. standard), and 0.150 mm (control sieve #100 U.S. standard) was washed with deionized water, and wiped dry with a lint-free wipes Kimwipes. After determining the mass of each sieve were installed at each other, starting with the smallest size (#100) at the bottom to the very large size (#8) at the top. In the top sieve slowly poured into 100 ml of the composition of the suspension gel of ha-VS/PEG-(SH)2with hyaluronic acid (Example 41). After a large part of the liquid component of the sample passed through the upper sieve, was slowly added to the top sieve of approximately 50 ml of 0.9% saline solution to wash out the component of the gel, which was detained on the sieve. After the liquid component is passed through the sieve, the sieve was removed from the stack. This process is repeated as long as each sieve was not washed and removed from the stack. Drop excess saline solution, which remained on each sieve was removed with toilet paper. Determined the total mass of each sieve (the weight of the sieve and the delayed gel). Expected mass of the gel particles detained on each sieve by subtracting the original weight of the sieve from the total mass of the sieve. Expected percentage of detained each sieve gel by dividing the mass of the gel, the detainee on a particular sieve, by total weight of the gel, the detainee on all screens.

Table 18
Size sieve #Size of sieve (mm)The gel retained on each sieve (%)
The Expo-
riment-1
The Expo-
riment-2
The Expo-
riment-3
AVERAGEThe STANDARD DEVIATION
82,362,22,52,02,30,3
141,40the 9.79,89,29,60,3
181,0033,229,127,229,83,1
200,8525,422,124,924,11,8
300,6014.4V19,119,1of 17.52,7
400,42510,08,510,39,61,0
600,253,4the 5.75,1the 4.71,2
1000,151,83,12,32,40,7

EXAMPLE 44

STERILITY AND TESTING FOR ENDOTOXIN

Gel GK-VS/PEG-(SH)2with GK (Example 42, Example 5) and gel GK-VS/PEG-(SH)2/TA and SC (Example 37, TA10) was tested for sterility and endotoxin using the company's WuXi AppTec using Protocol # BS210CY.203 and VESU.203, respectively. All samples were sterile and had levels of endotoxins <0,5 IU/ml

EXAMPLE 45

IN VIVO BIOCOMPATIBILITY TEST GK-VS/PEG-(SH)2With Ledger

To study the biocompatibility in vivo and pitamaha material in comparison with commercially available viscoelastic product was conducted the following in-vivo study in goats.

Used to study materials:

Subject material: Hydrogel - ha-VS/PEG-(SH)2with SC [Series NB51:119]

Reference material: Synvisc - manufactured viscoelastic Supplement

For this study used a total of 6 goats to form a skeleton. They were purchased from a source approved by the Ministry of agriculture of the United States. In the beginning of the study, the animals weighed between 65 and 99 pounds. Goats were tested for the absence of goat arthritis-encephalitis (CAE) and diseases of John, before they were put to the study. For each animal, a survey was conducted General health (including visual inspection of the limbs, nastrugannoy respiration, and absence of diarrhoea and nasal discharge) by a qualified veterinarian before you expose them to study. Animals were examined for any sign of disease or claudication. The selection of animals in the group for the study was determined by the absence of disease, clinical health, and lack of use of the knee joint research in the past. Goats were subjected to acclimatization during the relevant period of time defined by the relevant standards. Conditions of animal corresponded to deistvuyushchim and regulations, related to laboratory animals, namely, the Act on the protection of wild animals, Common law 89-544, as amended by the change in the General law 99-198, Federal register 52:16, the Ministry of agriculture U.S. Department inspection of animals and plants (USDA-APHIS), 1985 and Policy of the Ministry of health of the United States the humane treatment of laboratory animals, office of protection from risks when conducting scientific research/national Institute of health, USA, September, 1986. After injection of goats kept inside the premises of large pastures (paddocks). During the whole time the goats were in conditions of free-riding behavior. All animals received approximately 2 pounds a day diet for a small some animal, and unground hay. Access to piped water was unlimited. Feeding was stopped for approximately 12-24 hours before anesthesia, and access to water was stopped for approximately 12 hours prior to injection. Each animal was marked with a special ear tag.

Therapy

The study was planned as follows.

Table 19
Groups and assigned therapy
GroupEar tagRight stake is about
(1.5 ml)
Left knee
(1.5 ml)
The time of killing the animal after injection into the right knee injection in his left knee
1A3750The tested material
Hydrosalinity subject material
Reference material
Synvisc
24±1 hours
1A3751The tested material
Hydrosalinity subject material
Reference material
Synvisc
24±1 hours
1A3752The tested material
Hydrosalinity subject material
Reference material
Synvisc
24±1 hours
1B3597Reference material
Synvisc
The tested material
Hydrosalinity subject material
24±1 hours
1B3753Reference material
Synvisc
The subject mater is al
Hydrosalinity subject material
24±1 hours
1B3754Reference material
Synvisc
The tested material
Hydrosalinity subject material
24±1 hours
Total6

The main method of injection was the same for all subjects. All injections were performed under strict sterile. Animals were anestesiologi by intravenous injection of diazepam (0.1-0.5 mg/kg) and ketamine (4,4-7.5 mg/kg). Every knee was physically examined for the knockout, range of motion, swelling, temperature, crepitation, on the trajectory of the patella and valgus/varus violations. All injections were performed using conventional aseptic techniques. The left and right knee were prepared for injection by vystrogana wool in their respective fields and then disinfect them by careful treatment with chlorhexidine. The animal was transferred to the supine position. Right knee disinfected three times by careful treatment with chlorhexidine, alternating treatment with 70% isop epilogo alcohol, and stained with iodine solution.

For injections in each knee joint used a standardized methodology. A sterile needle 21 size length of 2 inches was introduced in the intra-articular space through the anteromedial access. Injected the needle until it touches the side wall mimimally cutting the medial femoral condyle and gently took her back. Were injected with the right joint, 1.5 ml of the test material for a Group 1A or 1.5 ml Control material for Group 1B. The injection needle was removed, and was supported by increased pressure in the injection. Injected knee then 20 times were subjected to a full cycle on "bending". Immediately after this, the left knee disinfected three times by careful treatment with chlorhexidine, alternating treatment with 70% isopropyl alcohol and stained with iodine solution, and were injected with left knee 1.5 ml Control material for Group 1A or 1.5 ml of the test material for Group 1B in the same manner as described above for the right knee. The injection needle was removed, and was supported by increased pressure in the injection. Injected knee then 20 times were subjected to a full cycle on "bending".

After the injection was tested those animals that showed signs of discomfort and dis is importa, and if necessary, they have introduced additional analgesic. All methods of treatment recorded in the relevant documentation on the study.

Animals were killed humanely at 24±1 hours after the start of injection by intravenous diazepam 0.22 mg/kg and ketamine 10 mg/kg to achieve a state of General anesthesia. Then shot the animals were injected intragastrically lethal dose of concentrated solution of potassium chloride (KCl) as long as not stated cardiac arrest.

Analysis

Visual morphological observations

After collecting the knee joints were opened and conducted are described in table 20 visual inspection injected knee joints. Spent photographing. Degenerative changes in the joints were not evaluated.

Table 20
Visual assessment and collection of samples
SampleVisual assessmentCollecting samplesPictures-varovanie and points
Synovial fluid (left and right)XX
Left and right the knee joints XX
The left and right synoviumXX

In addition, conducted a semi-quantitative estimation of the joint with a single expert, as shown in Table 21.

Table 21
Qualitative assessment based on visual inspection
PointsStainingHyperemiaSwelling
0NormalNoNo
1Slightly yellowLightLight
2YellowModerateModerate
3VisibleVisible

The total number of points when a visual inspection is amounted to the sum of the scores of staining, hyperemia, and edema (0-8 points).

Evaluation of synovial fluid

After collecting synovial fluid from open joints, which recorded the total volume. Visual and semiquantitative scoring was assessed viscosity, transparency and the color of the liquid, as shown in Table 22. Using hemocytometer determined the total number of leukocytes. In addition, a smear was prepared synovial fluid for the differential microscopic analysis. The remaining synovial fluid was kept frozen in cryovials with individual labels at -80°C. Smear synovial fluid was retained for possible future analysis.

Table 22
Description and scoring of synovial fluid
PointsColorTransparencyTendon
0S=STRAWC=TRANSPARENTN=NORMAL
1P=PINKH=WHITEA=ABNORMAL
2 Y=YELLOW R=REDD=CLOUDYW=WATERY
3B=BLOODYT=OPAQUE

The total number of points for synovial fluid was the sum of the scores of color, transparency and tendons (0-8 points).

Results

In the table below and in FIG. 14 shows that GK-VS/PEG-(SH)2since ha is biocompatible in the joint for 24 hours in experimental models of goats.

Table 23
Visual scoring of synovial fluid and joint (sorted relative to the test material (TM) and Control material)
Ear tagGroupThe test productThe scores for jointVolumeColor;
Transparency; tendon
The scores for synovial fluidTotal points
37501ATM0 1,6SHN11
37511ATM01,4SHN11
37521ATM01SHN11
35971BTM01,1SHN11
37531BTM00,75PHN22
37541BTM00,45SHN11
AVERAGE 0,01,11,21,2
The STANDARD DEVIATION0,00,40,40,4
35971BControl02,2SHN11
37531BControl01,9PHN22
37541BControl01,5SHN11
37501AControl01,3 SHN11
37511AControl00,8SHN11
37521AControl11,85SHN12
AVERAGE0,21,61,21,3
The STANDARD DEVIATION0,40,50,40,5
Color: S=straw (0), Y=yellow (2), P=pink (1), R=red (2), B=bloody (3)
Transparency: C=clear (0), H=white (1), D=dull (2)
Tendon (viscosity): N - normal (0), A=abnormal (1), W=watery (2)
TM=spacemy material

1B
Table 24
Differentiation of cells and the number of leucocytes (WBC) in synovial fluid
Ear
tag
GroupThe tested materialWBC/
mm3
The percentage of cells
in synovial fluid
The presence of
TM
TM
with over surface cells
Macro/mono
phagocytosis
TM
The absolute number of WBC
%
PMN
%
Lymph-city
%
Mono-city
%
Eosino-fily
%
Basic-fily
37501ATM23880820180 0++ v little+38224000
37511ATM72109001000++ v little+10094000
37521ATM7127892900++ v little+7127000
35971BTM69227741900++ v little+7614200
3753TM37897432300++ v little+2841750
37541BTM22006333400++ v little+990000
average852379,22,018,80,00,011148492
standard
deviation
7807the 10.11,7 9,20,00,013875201
37501AControl224097172200++ moderate+29131700
37511AControl7830875800++ many+6264000
37521AControl267007222600++ many49395000
35971BControl61307422400++ moderate+13486000
37531BControl54675354110++ many+10387300
37541BControl81005324500++ many+12150000
average 1277368,3the 3.827,70,20,020135667
standard
deviation
928013,22,113,50,40,016324705
(Sorted relative to the test material (TM) and Control material)
PMN=polymorphonuclear leukocytes
WBC=white cells
TM=the Tested material

For professionals in this field is obvious that the above described embodiments can be made without deviating from the General idea of the invention. Therefore, it should be borne in mind that the disclosed specific embodiments of do not limit the invention and it is intended that the invention covers the modifications are consistent with the essence and scope of the invention defined by the adoption of the report by the claims. Any combination of the above-described elements in all possible variations is included in the scope of the invention unless the description indicates otherwise, or unless it is clearly contrary to the meaning.

1. Hydrogel for use in medicine, formed by the reaction of hyaluronic acid with 1-10% of hydroxyl groups, derivatizing by reaction with diphenylsulfone ("2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid"), with cross-linking reagent, which represents a thiol-functionalized polyethylene glycol having 2 or 4 tirinya group.

2. The hydrogel under item 1 wherein the hyaluronic acid has a degree of conversion of hydroxyl groups in 2-(vinylsulphonyl)ethoxy group selected from 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

3. The hydrogel under item 2, where the hyaluronic acid has a degree of conversion of hydroxyl groups in 2-(vinylsulphonyl)ethoxy groups of approximately 4-5% of a recurring unit of the disaccharide.

4. The hydrogel according to any one of paragraphs.1-3, where hyaluronic acid is srednekamennogo molecular weight in the range from 700 to 3 million daltons.

5. The hydrogel according to any one of paragraphs.1-3, where the functionalized by thiol polyethylene glycol has a molecular weight in the range from about 250 to about 20,000 daltons.

6. The hydrogel under item 5, where the functionalized by thiol polyethylene glycol is the tsya polietilenglikolya (PEG-dithiol).

7. The hydrogel under item 1, where the functionalized by thiol polyethylene glycol is four and has Interative the kernel.

8. The hydrogel according to any one of paragraphs.1-3, 6 or 7, comprising less than ten percent of unreacted tylnej groups and less than 10% unreacted vinylsulfonic groups.

9. The hydrogel according to any one of paragraphs.1-3, 6, or 7, including the mass percentage of the polymer in water in the range from about 0.5 to 5.0 percent.

10. The hydrogel according to any one of paragraphs.1-3, 6 or 7, comprising a bioactive agent.

11. Composition for use in medicine comprising particles of a hydrogel according to any one of paragraphs.1-9 in an aqueous solution of hyaluronic acid.

12. The composition according to p. 11, where the hydrogel particles have a size in the range of approximately 0.10 to 3.0 millimeters.

13. The composition according to p. 11 in the form of a water suspension.

14. The composition according to p. 11, where the aqueous solution is a saline solution.

15. The composition according to p. 11, where the composition is a sterile composition.

16. The composition according to p. 11, further comprising a bioactive agent.

17. The composition according to p. 16, where the bioactive agent is a corticosteroid.

18. The composition according to p. 17, where the corticosteroid is a pharmaceutically acceptable salt of triamcinolone.

19. The composition according to p. 18, where the corticosteroid is triamcinolone acetonide.

20. Composers who s on p. 11, comprising living cells.

21. The composition according to p. 11, packaged in a syringe.

22. The hydrogel according to any one of paragraphs.1-3, 6, or 7, is packaged in a syringe.

23. The composition according to p. 21, extrudable through a syringe with needle 21 size.

24. The hydrogel according to p. 22, extrudable through a syringe with needle 21 size.

25. The use of hydrogel on one of the PP.1-10 for introducing into the intra-articular space of a joint of a subject.

26. The use of a composition according to any one of paragraphs.11-20 for introducing into the intra-articular space of a joint of a subject.

27. Application on p. 25 for the treatment of acute or chronic inflammation.

28. Application on p. 26 for the treatment of acute or chronic inflammation.

29. Application on p. 27, where acute or chronic inflammation associated with osteoarthritis, rheumatoid arthritis, other inflammatory arthritis and repetitive use.

30. Application on p. 28, where acute or chronic inflammation associated with osteoarthritis, rheumatoid arthritis, other inflammatory arthritis and repetitive use.

31. Applying a hydrogel according to any one of paragraphs.1-10 for injection or implantation near or inside the bones, teeth, nerves, cartilage, blood vessels, soft tissues or other tissues of a mammal.

32. The use of a composition according to any one of paragraphs.11-20 for injection or implantation near or inside the bones, teeth, nerves, HRA is a, blood vessels, soft tissues or other tissues of a mammal.

33. The way to obtain (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid, comprising (i) reaction of hyaluronic acid with a molar excess of diphenylsulfone at ambient temperature for a period of time up to 3 minutes, resulting in the interaction of from 1% to 10% of hydroxyl groups on the repeating disaccharide units of hyaluronic acid with diphenylsulfone.

34. The method according to p. 33 carried out in an aqueous solution of the base.

35. The method according to p. 33, where stage reaction is carried out at a temperature of 20-25°C.

36. The method according to p. 33, where stage reaction is carried out in a period of time from 10 seconds to approximately 120 seconds.

37. The method according to p. 34, where an aqueous solution of a base selected from an aqueous solution of sodium hydroxide or aqueous potassium hydroxide.

38. The method according to p. 34, where the method further includes interrupting the reaction by adding acid.

39. (2-(Vinylsulphonyl)ethoxy)1-10%hyaluronic acid for use in medicine, obtained by the method according to any of paragraphs.33-38.

40. Application (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid to obtain a hydrogel having a low degree of cross-linking reaction with a cross-linking reagent, which represents a thiol-functionalized polyethylene glycol having 2 or 4 tirinya group.

41. A method of producing a hydrogel having a low degree of crosslinking, comprising the reaction of (2-(vinylsulphonyl)ethoxy)1-10%hyaluronic acid with a crosslinking reagent, which represents a thiol-functionalized polyethylene glycol having 2 or 4 tirinya group in aqueous solution at a temperature in the range from 20°C to 45°C.

42. The method according to p. 41, where the reaction is carried out at physiological pH value.

43. The method according to p. 41, where the cross-linking reagent, which represents a thiol-functionalized polyethylene glycol, sterilized prior stage of the reaction.

44. The method according to p. 41, where after the reaction mixture allow to react for a period of time from 8 to 36 hours with the formation of the gel.

45. The method according to p. 41, carried out in sterile conditions.

46. The method according to any of paragraphs.41-45, additionally comprising the addition of active funds in an aqueous solution.

47. The method according to p. 46, where the active agent is a corticosteroid.

48. The method according to p. 47, where a corticosteroid selected from triamcinolone acetonide, hexacetonide triamcinolone, bentonite triamcinolone and puritania triamcinolone.

49. The method according to p. 47, where the corticosteroid is triamcinolone acetonide.



 

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: chemistry.

SUBSTANCE: invention relates to natural polysaccharide polymers and can be used in medicine. The obtained water-soluble bioactive nanocomposite includes a melanin compound-modified hyaluronic acid salt as a matrix and gold nanoparticles as filler. The method includes chemical reaction of solid-phase hyaluronic acid powder, a melanin compound, aurichlorohydric acid or a gold salt in conditions of simultaneous pressure action in the range of 50 to 1000 MPa and shearing deformation in a mechanochemical reactor at temperature of -18° to 110°C.

EFFECT: invention enables to obtain a water-soluble bioactive nanocomposite with high output of the end product and high content of gold.

4 cl, 18 ex

FIELD: chemistry.

SUBSTANCE: stabiliser includes modified chitosan which is obtained by modifying chitosan particles located in an emulsion of an organic solvent - water, with pH 6.0-6.5, by first reacting a mixture consisting of a carboxylic acid in an organic solvent and a condensing agent, and then with an organic base, wherein the carboxylic acid used is either palmitic acid or stearic acid or dodecanoic acid, the condensing agent used is a mixture of hydroxysuccinimide and an aliphatic carbodiimide or formaldehyde and an aliphatic isocyanide, and the organic base used is triethylamine.

EFFECT: effective liposome composition stabiliser which can be obtained using a simple method.

8 cl, 3 tbl, 5 ex, 7 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic synthesis. A method of obtaining a water-insoluble sulphur-containing chitosan-based biopolymer includes interaction of chitosan with a thiomethylating agent, preliminarily obtained by saturation of a formaldehyde solution with gaseous H2S, with molar ratio chitosan: formaldehyde: hydrogen sulphide 1:6:4, at a temperature of 60°C for 20-25 hours.

EFFECT: invention ensures obtaining the water-insoluble sulphur-containing chitosan-based biopolymer, which possesses a complexing activity to ions of noble metals (Pd, Pt).

1 ex, 1 tbl

FIELD: biotechnologies.

SUBSTANCE: skins of pond fish are flushed with cold flushing water during 10-15 minutes. They are crushed to the size of 2-3 mm. Water extraction is performed at the temperature of 40-45°C during 40-50 minutes at the ratio of crushed skins to water, which is equal to 1:1 at periodic mixing. Then, they are filtered; liquid fraction is dried in a spraying drier at the drier outlet product temperature of 60-65°C during 15-25 minutes so that hyaluronic acid is obtained. Solid fraction is subject to bleaching during 12 hours with hydrogen peroxide-salt solution that is prepared by mixing of 1 l of 3% hydrogen peroxide and 20 g of sodium chloride. Treatment of bleached solid fraction is performed with 1.0-1.2% solution of sodium hydroxide during 24 hours at the temperature of 20-25°C with further neutralisation of the obtained mixture with 3% boric acid solution. Treatment of swollen solid fraction is performed with Pancreatin ferment preparation solution taken in the quantity of 0.5-0.6% to the weight of solid fraction during 1.5-2.0 hours at the temperature of 37-40°C. Flushing of solid fraction is performed with cold flushing water for removal of Pancreatin residues so that collagen is obtained. The obtained collagen, depending on the purpose, is supplied for drying in drying chambers with forced air circulation at the temperature of 18-20°C during 12 hours and storage in dry ventilated rooms at the temperature of not higher than 20°C during 24 months or frozen to the temperature of minus 18 - minus 20°C and stored at the temperature of minus 18 - minus 20°C during 24 months. The liquid fraction dried in the spraying chamber is stored at the temperature of 0-4°C during 12 months or dissolved in physiological buffer solution.

EFFECT: improvement of the method.

2 dwg, 1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: production method of glucan-chitosan complex from yeast biomass of brewing waste involves mechanical and ultrasonic treatment of yeast biomass, destruction of proteins by treatment of the obtained suspension using alkali reagents with further extraction of a target product. As biomass, Saccharomyces living yeast is used. First, yeast is frozen to -15°C during 24 hours. After mechanical destruction, biomass is treated for 15 minutes at 20°C in an ultrasonic bath with frequency of an emitter of 35 kHz and voltage of 285 W. Biomass is acidified with chlorhydric acid till pH=5.5 and treated with ferment preparation in the amount of one pellet containing lipase - 3500 units of Ph.Eur., amylase - 4200 units of Ph.Eur. and protease - 250 units of Ph.Eur. per kilogramme of biomass in terms of dry substance; then, lipid components of yeast are removed. Fermentation is performed at t=20-29°C during 30-60 minutes. Destruction of proteins is performed at 55°C by means of a water bath during 60 minutes by treatment using 4% water solution of caustic soda at the ratio of yeast biomass and alkali, which is equal to 1:4. The medium is neutralised and hydrosol of glucan-chitosan complex is deposited by centrifugation during 10 minutes. The deposit is dried at t=55°C during 48 hours.

EFFECT: invention allows improving the quality of the obtained complex and its biological activity.

3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to production of hydroxyalkyl derivatives of polysaccharides. The method of producing 2,3-dihydroxypropyl chitosan involves reacting chitosan with glycidol in the presence of hydrochloric acid with ratio glycidol:chitosan:hydrochloric acid = (2-6):1:1, at room temperature until a gel forms. The mixture is then heated at 55-65°C for 12-14 hours and the reaction mass is treated with water. The mixture is then deposited, subjected to hot extraction with water-soluble alcohols or ketones and dried.

EFFECT: invention simplifies the method of production and output of the end product and improves sorption properties of the compound.

1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented: using benzophenanthridine alkaloid salts for preparing therapeutic agents for treating tumours, wherein the alkaloid salt is found in the form luteic, phosphatidic or hyaluronic acid, the benzophenanthridine alkaloid salt with phosphatidic acid or hyaluronic acid, and a based pharmaceutical composition for treating tumours.

EFFECT: what is shown is cytotoxic activity of the sanguinarine salts according to the invention at least twice increased in all studied tumour cell lines in relation to the chloride salt; it is suggested to be caused by higher absorption by the tumour cells.

12 cl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a recovered imidised biologically compatible polymer functionalised by an imide group. The above polymer is selected from the group consisting of polyethylene oxide, partially or completely hydrolysed by polyvinyl alcohol, polyvinylpyrrolidone, polyethyloxazoline, polyoxypropylene oxide block copolymers (poloxamers and meroxapol), polyethylene oxide and poloxamine copolymer, carboxymethyl cellulose and hydroxyalkylated cellulose, polypeptides, polysaccharides, carbohydrates, polysaccharose, hyaluronic acid, dextran, heparin sulphate, keratan sulphate, chondroitin sulphate, heparin, alginate, gelatin, collagen, albumin, ovalbumin, complex polyphosphoesters, polylactides, polyglycolides, polycaprolactones, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, polymaleic acids, polyamino acids, polyvinyl alcohol, polyvinylpyrrolidone, polyhydroxy cellulose, chitin, chitosan, and copolymers, ternary copolymers, or combinations or mixtures of the aforementioned materials. Also, the invention refers to a composition for a tissue adhesive, a medical device and a pharmaceutical composition.

EFFECT: invention represents additionally modified or functionalised imidised polymers.

9 cl, 2 ex, 20 dwg

FIELD: biotechnologies.

SUBSTANCE: fish roe is homogenised. Fish roe hydrolysis is carried out with a ferment preparation "Collagenase" in presence of an inhibitor for 10-12 hours. Chitosan is added to the produced hydrolysate at the ratio of 0.5-1.0:1.0. Components are mixed.

EFFECT: invention makes it possible to accelerate process of chitosan-nucleic complex production.

1 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: bioactive porous 3D-matrix for tissue engineering involves a resorbed partially crystalline polymer having a porosity of 60-80% and a pore size of 2 to 100 mcm. A biopolymer gel having a particle size of 30-100 mcm is incorporated into a portion of the pores. A polymer/gel ratio makes 99:1 to 50:50 wt %. The matrix is prepared by grinding a mixture of gel and polymer powder having an average particle size of 100 mcm, and the prepared mixture fills prepared moulds to be placed in a high-pressure chamber wherein the temperature is increased to 25-40°C first, and then the CO2 pressure is increased to 4.0-25.0 MPa. The system is kept in the above environment for 1 hour, and then the chamber pressure is discharged to an atmospheric one for 30-120 minutes; thereafter the temperature is decreased to a room value, and the patterns are removed.

EFFECT: ensuring flexibility of using the matrix in various organs and systems, no toxicity, higher ability to tissue regeneration stimulation, prolonged effect of biostimulation.

6 cl, 5 ex, 1 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: conduit wall is presented by a material of random micro- and nanofibres of a bioresorptive polymer of poly(ε-caprolactone), and the content is presented by a self-assembled nanostructured hydrogel of acetyl-(Arg-Ala-Asp-Ala)4-CONH2(PuraMatrix™) oligopeptide. The above conduit is implanted in a complex with the direct local delivery of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) genes to be introduced into the proximal and distal nerve segments, while the formed conduit is implanted into a nerve rupture, and its ends are fixed with epineural sutures.

EFFECT: invention provides a stimulating effect on the invasion of regenerative medullated fibres, on the recovery of motor and sensitive nerve function, and enables improving the effect of the recovery of the nerve structure and function after the extended ruptures.

4 cl

FIELD: medicine.

SUBSTANCE: claimed invention is aimed at manufacturing intraocular lens (IOL), for introduction of posterior eye chamber in form of PC Phakic lens. IOL is formed from hydrogel material, formed by cross-linked polymer and copolymer component. Lens includes UV chromophore, which is benzotriazole.

EFFECT: IOL hydrogel material usually has relatively high index of refraction and/or possesses desirable degree of protection against irradiation.

12 cl, 3 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine. Described is implant, which can be injected in subcutaneous or intracutaneous way in form of monophase hydrogel, which contains gel, obtained from cross-linked hyaluronic acid and one of its physiologically acceptable salts.

EFFECT: obtaining subcutaneous implant used for filling wrinkles and stimulation of epidermal cells and/or supporting mechanical properties of skin density and elasticity.

15 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular to method of obtaining form-preserving aggregates of gel particles, in which aggregates are held together by physical forces of non-covalent bonds, such as hydrophobic-hydrophilic interactions and hydrogen bonds. Method of obtaining form-preserving aggregates of gel particles includes introduction of preliminarily obtained suspension of gel particles in polar liquid, where gel particles have absolute electrochemical potential, into receiving medium, in which absolute electrochemical potential of gel particles decreases, which results in fusion of gel particles into form-preserving aggregate.

EFFECT: invention allows to obtain form-preserving gel aggregates in situ so that form of aggregate is determined by place of application.

49 cl, 35 ex, 11 tbl, 33 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, more specifically to biocompatible alginate systems with the delayed gelatinisation process. There are offered sets and compositions for making a self-gelatinised alginate gel containing sterile water-soluble alginate and particles of sterile water insoluble alginate with a gelling ion. There are offered methods for dosing self-gelatinised alginate dispersion for making the self-gelatinised alginate gel. The methods can include dosing the dispersion in an individual. There is offered the self-gelatinised alginate gel of the thickness more 5 mm and not containing one or more sulphates, citrates, phosphates, lactates, EDTA or lipids. There are offered implanted devices coated with the homogeneous alginate gel. There are offered methods for improving viability of pancreatic islets or other cell aggregate or tissue, after recovery and while stored and transported.

EFFECT: group of inventions provides creation of the alginate gelling system which contains alginate and the gelling ions with high biological compatibility; enables the gelatinisation process without pH variations, connected with the other systems, and requires minimum ingredients, thus provides variation of gelatinisation time and gel strength depending on the specific requirements.

62 cl, 11 dwg, 2 tbl, 27 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing biologically compatible gel which is thickened with cross-linked polymer by cross-linking a given amount of at least one biologically compatible natural polymer in a solution by adding a defined amount of cross-linking agent, an additional amount of polymer with molecular weight over 500000 dalton in a solution, in which the reaction mixture is diluted to reduce concentration of polymer in the solution, and the cross-linking reaction is stopped by removing the cross-linking agent.

EFFECT: gel and its use for separating, replacing or filling biological tissue or for increasing volume of such tissue, or supplementing or replacing biological fluid.

11 cl, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine. Particles of the viscoelastic material chosen from group, consisting of polysaccharides and their derivatives which are suitable for injection with gel particles having the size in a range from 1 to 5 mm at action of a physiological saline solution are described. An implant for increase of volume of the soft tissues, containing particles of the viscoelastic material chosen from group, consisting of polysaccharides and their derivatives where the basic volume of the specified particles represents the gel particles, suitable for injection and having the size in a range from 1 to 5 mm at action of a physiological saline solution, is described. The way of increase of volume of soft tissues at a mammal, including a human being, including subepidermal introduction in a place of a body of the specified mammal in which it is desirable to enlarge volume of soft tissues is described.

EFFECT: augmentation of volume of soft tissues at a mammal.

24 cl, 4 ex

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: medicine.

SUBSTANCE: invention concerns medicine, namely to reconstructive surgery, traumatology-orthopedy, maxillofacial surgery, stomatology and can be applied at osteo-plastic operations. For delivery of medical products immediately in a zone of defect and their prolonged influence in the centre of a lesion medicinal preparations are dissolved in a normal saline solution in a dose providing local effect, collagen-containing component is added to a solution in the ratio 9-20 g: 100 ml of a solution also admix with the carrier from dispersed allotransplants in the ratio of 1:1-3.

EFFECT: method allows lowering a dose necessary for reception of medical effect in 10 times, and also allows accelerating reparative processes in a defect zone.

3 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a set, containing calcium sulphate hemihydrates, pressed particles of calcium sulphate dehydrate, additionally containing one or more therapeutically, preventively and/or diagnostically active substances, and sodium-carboxymethylcellulose (Na-CMC) and a water medium, including water. The ratio R of sodium-carboxymethylcellulose and calcium sulphate in the set constitutes from 0.1 mg of sodium-carboxymethylcellulose (calculated as Na-CMC)/g of calcium sulphate to 8 mg of sodium-carboxymethylcellulose (calculated as Na-CMC)/g of calcium sulphate. When mixed, the said components of the set form a bioresorbable ceramic composition. The invention also relates to the application of the set for the treatment of a disease or condition, associated with prostate. Also claimed is a composition ready for application in the form of a paste for introduction to a patient during the time period from 5 minutes to 1 hour before hardening, obtained by mixing the components of the set. Also claimed are: a hardened composition and a method of obtaining the hardened composition or the composition ready for application.

EFFECT: control of the time of the set and composition hardening.

13 cl, 12 tbl, 9 ex

Up!