Composition with bound hyaluronic acid for developing tissues

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

 

Cross references to related applications

This application claims the priority of practical application US registered December 22, 2003 (application number not yet assigned, attorney number in the list of cases for hearing 1769.2001-000), the full contents of which are incorporated here by reference.

The technical field to which the invention relates.

The invention relates to pharmaceutical compositions containing crosslinked hyaluronic acid, to methods for their preparation and use for building tissues and/or drug delivery.

The level of technology

Currently, all fillers to build tissues, approved by the Department for sanitary supervision of food and drug administration (USA), are derived collagen. Approximately 3-5% of people are serious allergic reactions to bovine collagen, requiring, therefore, thorough testing before use of such fillers for each specific subject.

Hyaluronic acid, also referred to as "GC", is present in nature water-soluble polysaccharide, which is the main component of the extracellular matrix and which is widely distributed in animal tissues. The molecular weight of ha of natural origin, mainly, is within approximately 6×104to approximately 8×10 6daltons. It has excellent biocompatibility and does not cause the reaction to a foreign body or allergic reactions after implantation of the entity.

Methods of obtaining commercially available hyaluronan well known. Known also various ways of connecting the Ledger and bound Ledger to reduce the water solubility and the ability to diffusion of GA and for increasing the viscosity of the Ledger. For example, see U.S. patent No. 5356883 and 6013679, the full meaning of which is incorporated here by reference. Further, many forms of CC are involved, for example, as surgical material to prevent postoperative adhesi tissues, as a Supplement to the synovial fluid in the joints, as a liquid substitute and/or surgical vspomoschestvovanyya in eye surgery, as a framework for the design of in vitro tissue or directed regeneration or expansion of tissues in vivo, etc.

The use of such products CC suffers from several drawbacks, such as, in particular, a compromise between the properties in vivo and applicability in surgery. For example, the Ledger, which is chemically modified or crosslinked to obtain the desired mechanical properties and bistability in vivo, may be so viscous that its injection through a thin needle is difficult or impossible. In contrast, GC, suitable for injection, may have in vivo lower bistabil the awn or mechanical properties.

Further, at present there is great interest to the means of delivery with a gradual release of biologically active agents, including, for example, therapeutic agents or drugs and biological samples. The most difficult task is the development of means of delivery, which can provide the desired level of bioavailability of therapeutic agent within a range to achieve the desired clinical result, if also desired balance between mechanical properties and bistability in vivo and applicability in the surgery/treatment. The bioavailability of a drug depends on the nature of the drugs used means of drug delivery and the delivery path, for example, oral, topical, subcutaneous, through the mucous membrane, the purpose of injection, the purpose of inhalation or the appointment of a combination of two or more of such ways. Bioavailability may be low as a result, for example, dissolution of drugs in the process of metabolism, fast or uneven collapse of the means of delivery, fast or uneven release of the drug from delivery vehicles, etc. may be accompanied by similar problems in the frequency of drug administration, difficulty of assignments, for example, difficulties injection, biodegradation, etc. in Addition to the difficulties noted above, frequent purpose nedostatocnosti of drug delivery may result in variations in drug delivery, resulting in increased number of cases of harmful side effects, reduce therapeutic benefit, etc.

Therefore, there is a need for compositions with SC, which overcome or minimize the above problems.

Disclosure of inventions

The invention is directed to compositions of hyaluronic acid (ha) and the methods of preparation and use of compositions Ledger that are effective for building tissues and/or drug delivery.

The composition of hyaluronic acid (ha) includes particles of gel crosslinked water-insoluble hydrated Ledger. The Ledger includes products knitting, represented by the following structural formula:

CC'-U-R2-U-HK',

where each GK' is the same or different crosslinked molecule Ledger', that is, the stitching can be intramolecular or intermolecular.

Each U is independent optionally substituted o-acyl isomotion or N-acyl urea.

R2is optionally substituted alkyl, alkenyl, quinil, alkoxy, cycloalkyl, cycloalkenyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cyclo-aliphatic alkyl, aralkyl, heteroalkyl or heterocyclic-alkyl.

Method of increasing tissue in a subject in need of building fabric, includes the step of introducing the subject of the needle in the place where you want to build tissue, and the needle is connected with a syringe is m, filled composition Ledger. Also included is the application of force to the syringe so as to deliver at the place of destination at least one portion of the composition Ledger.

The method of obtaining the composition of the group includes the formation of particles insoluble in water, dehydrated crosslinked ha, separation of particles insoluble in water, dehydrated crosslinked state average diameter, selecting a subset of the particle mean diameter and hydrating the subset digidrirovanny particles using a physiologically compatible aqueous solution, forming, thus, the composition of the Ledger.

Another way of preparing the compositions stitched Ledger includes stitching predecessor stitched CC bis-carbodiimide in the presence of a pH buffer with a pH between about 4 and about 8 and the de-hydration stitched GL with getting dehydrated crosslinked Ledger.

The method of stabilization stitched Ledger includes hydrating water-insoluble dehydrated crosslinked Ledger using physiologically compatible aqueous solution, forming, thus, a stable composition of the Ledger, where physiologically compatible aqueous solution includes at least about 0.1% by weight of the local anesthetic, where the value of the elastic modulus G' for the stabilized composition is at least about 110% of the value of G' for the unstabilized composition and when measured at 37°C and a frequency of 1 Hz using a flat geometry on 4 see

Stabilized composition Ledger is also included.

Embodiments of disclosed here, it is effective for the production and application of the gel particles crosslinked water-insoluble hydrated Ledger, where the stitching in the General Ledger include the connecting group R2that have the combination of biostability and mechanical properties in vivo, at the same time having improved workability, for example, improved ease of insertion through a thin needle. For example, as shown in the Examples section below, the composition of GC have improved values of the elastic modulus G' and a kinematic viscosity, at the same time showing improved biologist to the enzyme hyaluronidase in vitro and in vivo. Disclosed effective to apply a stitched CC to build tissues, though the frequency of implantation is reduced. Exercise is also effective to apply a stitched CC as a means of drug delivery, which demonstrates the surprising and unexpected effect of increasing biostability together with properties effective for the liberation of medicine, and the effective properties of the destination.

Brief description of figures

Figure 1. Absorption in the UV products matching obtained by the interaction of GC and p-phenylene-bis(ethylcarbodiimide) at the ratio of molar equivalents of 75%, 100% and 125%.

Figure 2. Absorption in the UV cont the mswb stitching, obtained through the interaction of GC and p-phenylene-bis(ethylcarbodiimide) in MES buffer at pH 5.5, 6.0 and 6.5 in.

Figure 3. The effect of particle distribution on the average diameter of the elastic modulus (G') of the gel.

Figure 4. The influence of the distribution of particles in the gel according to the average diameter of the force required to eject the gel from needle gauge/size 30.

Figure 5. Degradation of cross-linked product of GC in the presence of the enzyme hyaluronidase compared with other products to increase tissue: Restylane®, Perlina® and Hylaform®.

Figure 6. Profile of degradation of the gel with different initial G', obtained from cross-linked Ledger of the invention.

Figure 7. The elastic modulus G' and the degradation profiles of the gels obtained in phosphate buffer without lidocaine in buffer containing 0.2% lidocaine and 0.3% lidocaine.

The implementation of the invention

The invention relates to compositions with crosslinked ha, their preparation and methods of use thereof.

Unstitched Ledger, for example, the predecessor stitched CC of the invention, generally includes units/units of disaccharide consisting of D-glucuronic acid (Gcuk) and K-acetyl-O-glucosamine (Glcac), which are connected with opposite sides with the formation of the linear polymer. GC is often found in nature in the form of sodium salt, of sodium hyaluronate. Ha, sodium hyaluronate and drugs or Ledger or of sodium hyaluronate often is remembered as hyaluronan. As used here, the terms "GC" and "hyaluronan" also apply to any other salt of hyaluronic acid, including, for example, potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate, etc. Unstitched Ledger used as a precursor to knitting, usually has an average molecular weight in the range of from about 6×104to approximately 8×106Daltons, or from 150 to 20000 repeating units of the disaccharide. As starting material can be applied Ledger from any among a variety of sources, including General Ledger, extracted from animal tissues or obtained as a product of bacterial fermentation. On the other hand, unstitched Ledger used to obtain the compositions of the invention can be produced in industrial quantities at Bioprocess technology, as described in West et al., PCT Publication No. WO 86/04355, the full meaning of which is included here by reference.

Stitched CC can be obtained by reaction unstitched Ledger with a crosslinking agent under suitable reaction conditions. Stitched CC receive as a result of the seamless interaction of GC with bis-carbodiimide in the presence of a pH buffer, where the pH of the buffer is between about 4 and about 8. The pH value of the buffer can be between about 4 and about 7, typically between about 5 and about 6.5, and more usually between about 5 is approximately 6. In the preferred implementation a pH of about 5.5.

The term "pH buffer" may include any buffer substance known in the prior art, such as 2-(N-morpholino)-econsultancy acid (MES); 2,2-bis(hydroxymethyl)-2,2',2"-nitrotriazole; succinate/succinic acid; KH2PO4; N-Tris(hydroxymethyl)-2-aminoethanesulfonic acid; triethanolamine; diethylbarbituric; Tris(hydroxymethyl)aminoethane; N-Tris(hydroxy)methylglycine and N,N-bis(2-hydroxyethyl)glycine. The buffer compound can be applied with additional acid or base, such as 2-(N-morpholino)-econsultancy acid with NaOH; 2,2-bis(hydroxymethyl)-2,2',2"-nitrotriazole with HCl; succinate to succinic acid; KH2PO4with brown; N-Tris(hydroxymethyl)-2-aminoethanesulfonic acid with NaOH; with triethanolamine HCl, diethylbarbituric with HCl; Tris(hydroxymethyl)aminoethane with HCl; N-Tris(hydroxy)methylglycine with HCl; N,N-bis(2-hydroxyethyl)glycine with HCl. Preferably, the buffer comprises 2-(N-morpholino)-econsultancy acid and NaOH.

Typically, the buffer substance is dissolved in water at a concentration of between about 5 mm and about 250 mm, typically between about 10 mm and about 150 mm, more typically between about 25 mm and about 100 mm, and preferably about 75 mm.

Usually unstitched Ledger stir in the aquatic environment, such as solution pH-b is Fehr at a concentration of between about 1 mm and about 100 mm, typically between approximately 10 mm and approximately 50 mm, more typically, between about 25 mm and about 50 mm, preferably about 37 mm. Use an individual concentrations may vary depending on the molecular weight unstitched predecessor Ledger. At lower concentrations the reaction may slow down. At higher concentrations with the product may be more difficult to be managed by increasing its viscosity. Examples of acceptable concentrations unstitched Ledger for other crosslinking reactions are described in US patent No. 5356883 Kuo et al., the content of which is incorporated here by reference in its entirety.

The reaction can be conducted at a temperature in the range between approximately 0°and approximately 60°C., usually between about 10°and about 40°C., more typically between approximately 15°and approximately 30°, and preferably about 25°C. Examples of reaction conditions can be found in Examples 1-9.

Bis-carbodiimide can be combined with a solution of unstitched Ledger alone or more typically in the form of a solution in mixing with water, an organic solvent, for example acetone, methylethyl-ketone, dimethylformamide, dimethyl sulfoxide, methanol, ethanol, 2-propanol, acetonitrile, tetrahydrofuran, N-methyl-pyrrolidone and the like, More typically, the solvent is acetone and bis-carbodiimide on titsa at a concentration of between approximately 0.1 mg/ml and about 100 mg/ml, usually between about 1 mg/ml and about 50 mg/ml, more typically, between about 5 mg/ml and about 25 mg/ml, and preferably about 15 mg/ml

Unstitched CC and bis-carbodiimide you can join at any ratio of molar equivalents, for example between about 1% and about 200%, typically between about 10% and about 150%, more typically between about 18% and about 125%. In various implementations, the ratio of molar equivalents of approximately 18%, or approximately 38%, or about 50%, or about 75%or about 100%, or about 125%.

Composition Ledger made using bis-carbodiimide may include stitching, where the linking group, R2 bis-carbodiimide agent included in the stitching, for example, the linking group linking via a group of U at each end with a molecule Ledger', as shown in the following structural formula:

CC'-U-R2-U-GK'

Each Ledger' in the formula above may be the same or a different molecule Ledger, that is, the stitching can be intramolecular or intermolecular stitching. Each U may be the same or different and is an optionally substituted N-acyl-urea or O-acyl-samochowiec, as shown in the fragments in brackets in the following structural formulas:

These crosslinking can be formed in the reaction Ledger with cross-linking reagent bis-carbodiimide represented by the following structural formula:

R1-N=C=N-R2-N=C=N-R1,

where two carbodiimide, Deputy R1are connected through a linking group, R2.

In the foregoing structural formulas each R1may be the same or different and is an optionally group-Deputy, selected among hydrogen, aliphatic hydrocarbon (for example, alkyl, alkenyl, quinil), alkoxy, cycloaliphatic hydrocarbons (for example, cycloalkyl, cycloalkenyl and cycloalkyl), aryl, heteroaryl, heterocyclyl, of cycloaliphatic alkyl, aralkyl, heteroalkyl, geterotsiklicheskikh etc. possible Suitable substituents are those which do not change significantly the properties of the resulting composition stitched Ledger; they are described here in the section describing each of the relevant groups. In other implementations R1is optionally substituted aliphatic group. More preferably, R1represents alkyl, for example linear or branched alkyl, C1-C6such as methyl, ethyl, propyl, butyl, 2-propyl, tert-butyl and the like, Preferably, each R1is ethyl.

Each R2- this is when you need the ti substituted linking group, comprising one or more aliphatic, cycloaliphatic groups, aryl, heteroaryl, heterocyclyl, cycloaliphatic alkyl, aralkyl, heteroalkyl, heterocyclyl-alkyl, etc. possible Suitable substituents are those which do not change significantly the properties of the resulting composition stitched Ledger; they are described here in the section describing each of the relevant groups. R2can optionally include or be interrupted by another group, such as carbonyl, amide, hydroxy, sulfide, disulfide, etc. In other implementations R2is a cycloaliphatic, aryl, heteroaryl or heterocyclic group. In still other implementations R2is 1,6-hexamethylene, octamethylene, decamethrin, dodecamethyl, PEG, -CH2CH2-S-S-CH2CH2-, para-phenylene-S-S-para-phenylene, meta-phenylene-S-S-meta-phenylene, meta-phenylene or para-phenylene. More preferably, R2is phenylene. Preferably, R2is para-phenylene.

In one implementation, bis-carbodiimide selected from 1,6-hexamethylene-bis(ethylcarbodiimide), 1,8-octamethylene-bis(ethylcarbodiimide), 1,10-decametre-bis(ethylcarbodiimide), 1,12-dodecamethyl-bis(ethylcarbodiimide), PEG-bis(propyl(ethylcarbodiimide)), 2,2'-Dityatin-bis(ethylcarbodiimide), 1,1'-dithio-para-phenylene-bis(ethylcarbodiimide) and 1,1'-dithio-meta-phenylene-bis (ethylcarbodiimide is Yes). In the preferred implementation of the bis-carbodiimide is para-phenylene-bis(ethylcarbodiimide). Methods of obtaining bis-carbodiimides described in patents US№№6013679; 2946819; 3231610; 3502722; 3644456; 3972933; 4014935; 4066629; 4085140; 4096334; 4137386; 6548081 and 6620927, the contents of which are incorporated here by reference in its entirety.

It is believed that the PS reaction with cross-linking reagent bis-carbodiimide in the presence of available proton at the first stage involves protonation. The acido-anion/anion acid can then join the carbon atom forming cation, which leads to the formation of the intermediate O-acyl-isomotion. Acyl group of the intermediate can be moved from the oxygen atom to the nitrogen atom with the formation of a derivative of GK with N-acyl-smokeview. It is believed that the transition from oxygen to nitrogen may be incomplete, resulting in a mixture of products, including N-acyl-urea and O-acyl-smokeview. Thus, cross-linking, resulting in the reaction of bis-carbodiimide with the predecessor stitched Ledger, may typically contain two O-acyl-isomotion connected via R2as shown in the following structural formula:

or O-acyl-isomotion and N-acyl-urea, United through R2as shown in the following structural formula:

or two N-acyl-urea, the connection is built through R 2as shown in the following structural formula:

.

Mixed products can be used separately or together to obtain the compositions according to realizations of the invention.

Stitched CC you can besiege, pouring in miscible with water, an organic solvent, for example acetone, methylethyl ketone, dimethylformamide, dimethylsulfoxide, methanol, ethanol, 2-propanol, acetonitrile, tetrahydrofuran, N-methyl pyrrolidone and the like, preferably an alcohol, for example ethanol. The precipitate can be collected and dried, for example under reduced pressure.

Dry sewn Ledger can be made into particles by any method well known in this field, for example by abrasion, grinding, tearing and the like, preferably by grinding at cryogenic mill. On the other hand, not sewn Ledger can be planted during cooling (cryoprecipitate) with the formation of small particles, which can then be dried, or not stitched CC you can crush cryogenic mill and the resulting particles are then dried.

As used here, "water-insoluble" and similar terms refer to compositions, for example, insoluble in water digidrirovannye particles or water-insoluble hydrated particles that are heterogeneous at suspendirovanie in sufficient quantity of water at ControlTemplate. In one implementation of the "water-insoluble" means that once the particles of the composition of ha in water at neutral pH and 25°C is not less than approximately two weeks of particles Ledger substantially insoluble, that is essentially the group of particles is not freely dissolved in water. In other implementations "insoluble in water" means that the group of particles are essentially insoluble after not less than approximately 4 weeks in the above described conditions, usually after at least about 6 weeks, more typically, after not less than approximately 8 weeks or preferably after at least about 12 weeks. In one implementation of the "water-insoluble" means that the group of particles are essentially insoluble after not less than approximately 26 weeks in the above described conditions. As used here, "readily soluble" means dissolution of ha molecules in water separately from hydrated swollen particles in the water.

Moreover, derivatives stitched CC can be a hydrogel. As the term is used here, a "hydrogel" is a crosslinked macromolecular network, which can swell in water or biological fluids and retain a significant portion of water within its structure without dissolving. As used here, the term "swelling" refers to the intake of fluid, such as water, gel increase usually p and the application of heat or pressure. Hydrogels have a high molecular weight, which, usually, cannot be measured by conventional means, and are composed of polymer chains and links.

Particles of crosslinked Ledger can be characterized by the distribution of particle diameter. The average diameter can be measured as the average diameter of hydrated particles and/or the average diameter digidrirovanny particles. Typically, the average particle diameter is chosen from the group consisting of the average diameter of the hydrated particles between approximately 20 μm (micrometer) and approximately 1000 μm and average diameter digidrirovanny particles between approximately 10 microns and approximately 500 microns. In other embodiments, implementation of the average diameter of hydrated particles is between about 40 μm (micrometer) and about 600 μm, and the average diameter digidrirovanny particles is between about 20 μm and about 300 μm, or more preferably, the average diameter of hydrated particles is between about 50 μm (micrometer) and approximately 500 μm and the average diameter digidrirovanny particles is between about 25 microns and about 250 microns.

In a separate embodiment, the composition of the group of companies consists essentially of a hydrated gel particles crosslinked water-insoluble Ledger. For example, the composition of the Ledger this exercise you shall rassmatrivati as the only phase of the hydrated particles, i.e., any liquid in the composition is essentially contained in the hydrated particles, i.e., essentially free of liquid phase no. In other implementations, where the Ledger in composition, consists essentially of the crosslinked water-insoluble gel particles, certain forms of CC exclude from the composition, for example, normally exclude particles or molecules Ledger with an average diameter of less than about 1 μm, more typically exclude particles or molecules Ledger with an average diameter of less than about 10 microns, and preferably eliminate particle or molecule of ha with an average diameter of less than approximately 20 microns.

As used here, a "subset" of particles with an average diameter means that the sample particles characterized by an average diameter and at least some fraction of the particles rejected, that is not include in the subset.

In other implementations, the average particle diameter are selected, for example, by sieving or by other methods well known in the field, so you can select a specific diameter distribution to obtain special properties of the final composition, for example, as shown in the Examples section below. Thus, digidrirovannye particles can be sifted to separate fractions by the average size of, for example, as in Example 16, which collect five factions at the average diameter of the particles possess: 0-25 μm, 25-75 μm, 75-125 microns, 125-180 180-250 microns and microns.

Different fractions according to the average diameter of the individual particles of the composition can be used to specify combinations of fractions at an average diameter in different proportions, which will lead to a special combined properties. Thus, in one implementation insoluble in water digidrirovannye particles can be separated by not less than two fractions according to the average diameter and fractions can be combined in such proportions as to adjust the properties of the combination, for example in example 17 two fractions containing particles of 125-250 microns and 0-125 μm, are combined in a ratio of 1:1. The resulting composition has a distribution diameter than that of the original particles to sort by size, for example, the distribution of the diameter can be multimodal distribution by average diameter, for example a bimodal distribution mean diameter when the compositions choose two fractions according to the average diameter. Properties of multimodal compositions are built from the properties of the individual fractions of the average diameter and their quantities in the composition. In another implementation insoluble in water digidrirovannye particles can be separated by not less than three fractions according to the average size of the particles, where at least two fractions according to the average diameter is chosen and at least one fraction by the average d is ametro drop.

Digidrirovannye particles usually hydratious in the presence of a physiologically acceptable solution (for example, physiological saline or phosphate buffer, as described in the Examples section) under conditions comprising a temperature of at least about 100°C., a pressure of at least approximately 120 kPa (kilopascals) and the duration of at least approximately 15 minutes Such conditions can be achieved in an autoclave, for example, also be used for sterilization of particles. Other conditions include a temperature from about 100°to about 150°C., usually from about 110°to about 140°C., or preferably from about 120°to about 140°C.; a pressure of about 120 kPa to about 200 kPa, usually from about 120 kPa to about 160 kPa, or preferably from about 130 kPa to 140 kPa; duration of from about 15 to about 75 minutes, more typically from about 20 minutes to about 60 minutes

Additional review sterilization methods/operations include the following. In one implementation, the product can be exposed pure steam at a temperature of 118-133°C (typically, approximately 121°C) and the corresponding vapor pressure at saturation (from about 103 kPa to approximately 186 kPa). Ohlord is the evaporation may occur during vacuumization or natural cooling can occur for example, in the slow removal of the vapor system pressure. In another implementation, the product can be sterilized at a temperature of 118-133°C (typically 121°C), using a mixture of air with vapor, preferably circulating, for example, under the fan. Normal pressure is 69 kPa to about 103 kPa above the saturated vapor pressure (usually less than 310 kPa). Cooling can be achieved, for example, the introduction into circulation under the fan cold air. In another implementation, the products can be sprayed with liquid water at a temperature of sterilization, if enough pressure on the meter to keep water in a liquid state (usually less than 310 kPa). Cooling to about 80°C can be achieved by direct contact of the water spray with the cooling system in the heat exchanger. Cooling from approximately 80°to approximately 20°C is mainly due to evaporation and may serve to remove from the product of the excess fluid. There are many variants of the above-described techniques hydration/sterilization.

The composition is crosslinked Ledger preferably comprises a physiologically effective amount of at least one bioactive agent selected from the group consisting of cells, genes, proteins, antibodies, peptides and pharmaceutical substances.

"Drug substance" as the term is used here, which concludes, for example, the compounds and compositions recognized in the official United States Pharmacopeia, official homeopathic Pharmacopoeia of the United States, or official national formulary (National Formulary or any Supplement to them, the compounds or compositions intended for use in diagnosis, cure, mitigation, treatment or prevention of disease in humans or animals, and compounds and compositions (other than food)intended to affect the structure or any function of the body of man or animals. Pharmaceutical substances include pharmaceutical compounds and solutions, salts, crystalline polymorphs and their stereoisomers. Examples of classes of pharmaceutical substances include growth factors (e.g., interleukins, prostaglandins, thromboxanes, leukotrienes, and cytokines), steroid and non-steroid contraceptives, antibiotics (such as penicillin, streptomycin and lincomycin), analgesics, anesthetics, sedatives, barbiturates, aminoalkylsilane, catecholamines, drugs, antagonists drugs, antineoplastics agents and anticoagulants (eg, heparin sulfate and heparin), steroidal and non-steroidal anti-inflammatory drugs, anesthetics, antibiotics, antifungal agents, hormones, and any synthetic analogues and farmaceuticas and active fragments of the above, etc. Pharmaceutical substances suitable for use in the delivery systems in the implementation of the invention may be fat-soluble, water-soluble, anionic or cationic until they are able to react with group a derivative of hyaluronic acid with the formation of covalent or ionic bonding or hydrophobic or hydrophilic interactions, for example in the implementation may be a hydrophobic interaction between the hydrophobic part of the pharmaceutical compounds and derivatives Ledger.

The bioactive agent can be introduced at any stage, but usually it is added during the preparation of compositions by the incorporation of physiologically compatible solution used for hydrating digidrirovanny particles, for example phosphate buffer in the examples. In one implementation, the composition is crosslinked Ledger, for example hydrated particles Ledger includes an anesthetic, such as a local anesthetic as a bioactive agent. A local anesthetic may include at least one member selected from amburana, amalananda, amylocaine, benoxinate, benzocaine, ethoxyquin, bifrenaria, bupivacaine, butacaine, butamben, butanilicaine, bumetamide, butokukan, Kartikeya, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, timesonline, dimethocaine, diperodon, dicyclomine, ecgonidine, e is gonina, ethylchloride, etidocaine, beta-eucaine, eufrocina, phenalkamines, formosana, hexylcaine, hydroxytyramine, isobutyl-p-aminobenzoate, latinateen, mesylate, leokadya, lidocaine, mepiwakaina, Marikina, metabolisation, methyl chloride, myrtecaine, nahapana, octocaine, articaine, oxethazaine, paradoxicon, phenacaine, phenol, piperocaine, pyridoxine, polidocanol, pramoxine, prilocaine, procaine, proparacaine, proparacaine, propiomazine, propoxycaine, pseudococaine, procaine, ropivacaine, salicylic alcohol, tetracaine, topicaine, trimekainom, Salamina and their salts. Typically, the bioactive agent is a local anesthetic selected from lidocaine, mepiwakaina, prilocaine, bupivacaine, cocaine, procaine, chloroquine and tetracaine or their salts or solutions. Preferably, the bioactive agent is a local anesthetic lidocaine or its solution or salt, such as salt lidocaine ·HCl.

Stitched CC can serve as a tool that provides a controlled or sustained release of the bioactive agent. In one implementation GK controlled-release was placed in contact with the pre-selected tissue and left in place to achieve the desired clinical result. GK controlled-release version of the implementation, you can enter through inye the tion or implant at the site, where desirable, shipping, or you can assign through the mouth or the way, which is a combination of two or more of these ways purpose.

Diffusion provides for the delivery of the bioactive agent through the delivery system, in which the drug reacts with stitched CC is not covalently. Such non-covalent interactions include ionic, hydrophobic and hydrophilic interactions, in which the bioactive agent is dispersed within the carrier. As used here, the term "dispersed" means an ionic, hydrophobic and hydrophilic interactions between medicine and the state.

The speed of delivery of the bioactive agent is associated not only with the speed of its diffusion, but also with the rate of degradation of the Ledger, in which a drug or other bioactive substance is dispersed. The rate of degradation of the Ledger associated with the degree of crosslinking and also depends on many metabolic processes in vivo. The degradation process is usually slower diffusion. By selecting the concentration of the drug dispersed within the group, and the degree of crosslinking can be controlled rate of degradation and diffusion and thus the rate of drug delivery.

As used here, "physiologically effective amount" is the amount of bioactive agent, which is sufficient for the desired effect, for example, the number of local an statica, sufficient for anesthetic action on the subject, which made the injection composition comprising the agent. Experienced in this field will be able to determine physiologically effective amount, based on the amount of the applied composition, the medical history of the subject, etc. are the Concentration of drug can be varied within very wide limits and preferably should be chosen depending on the degree of crosslinking of the Ledger, the solubility of the drug, its pharmaceutical activity and the desired actions.

As used here, a "physiologically acceptable solution is any solution known in this field, which is used as a carrier in physiological systems, such as aqueous solutions, which are usually sterile, allergeni, non-toxic and the like, such as saline solution, buffer solution, sugar solution, etc.

Viscoelasticity properties of the composition can be defined as shown in the Examples. In one implementation, the composition has at least one parameter measured at 37°C, selected from the elastic modulus G' of not less than about 50 PA (Pascal), measured at a frequency of 1 Hz (Hertz) using a flat geometry 4 cm; and a kinematic viscosity of not less than about 20,000 CPS (centipoise)measured at shear rate 1 s-1.

In another implementation of kinematics what I viscosity is not less than approximately 40000 SP, more typically less than about 60000 SP, and preferably not less than approximately 70000 SP. In another implementation of the kinematic viscosity is from about 20,000 CPS to about 250,000 in the JV. In other implementations kinematic viscosity is from about 40000 SP to approximately 220000 JV, more typically from about 60000 SP to approximately 200000 JV, and preferably from about 70000 JV to approximately 170000 SP.

In other implementations, the elastic modulus G' is at least approximately 100 PA, typically not less than about 100 PA, more typically, no less than about 200 PA, and preferably not less than about 400 PA. In other implementations, the elastic modulus G' is from about 50 PA to about 1,600 PA, typically, from about 100 PA to about 1200 PA, more typically from about 200 PA to about 1000 PA, and preferably from about 400 PA to about 700 PA.

Composition with stitched CC can be characterized by bistability, that is resistant to degradation in vitro by the enzyme hyaluronidase, as shown in the Examples. For example, when the connection of the composition at 37°C with the enzyme hyaluronidase in the amount of approximately 0.3% by weight under conditions suitable for hyaluronidase reaction, the value of G' is La song measured after 16 h the reaction is not less than approximately 5% of the value of G'measured after approximately 15 min from the beginning of the reaction. In other implementations, the value of G' for the composition measured after approximately 16 h of reaction, is the portion of the value measured after approximately 15 min from the beginning of the reaction, generally, not less than about 10%, or not less than about 25%, or not less than about 45%, or not less than about 50%, typically not less than about 60%, more typically no less than about 75%, or preferably not less than approximately 80%. In one implementation, the value of G' for the composition measured after approximately 16 h of reaction, approximately the same as measured after approximately 15 min from the beginning of the reaction.

In other implementations, the elastic modulus G' is increased, that is, the composition is stabilized by incorporating a local anesthetic, such as lidocaine, compared with the unstabilized composition, i.e., identical composition, except that a local anesthetic is not included. For these accomplishments stabilized and unstabilized compositions can be compared by measuring the value of G' in the same conditions. A stable composition is obtained by hydrating particles in terms of hydration, RAS is rytych here by using a solution containing 0.1%local anesthetic (eg, lidocaine) in weight, has a higher G'than G' at the unstabilized composition, which is usually at least about 110%, typically less than about 120%, more typically less than about 150%, and preferably not less than approximately 175%.

In a separate implementation of the composition with the Ledger includes hydrated particles of gel crosslinked water-insoluble Ledger. Particles include lidocaine ·HCl. And the particles have an average diameter selected from the group consisting of hydrated particles of average diameter between about 20 and about 1000 microns and digidrirovanny particle average diameter between about 10 and about 500 microns. Next, the particles include knitting, represented by the following structural formula:

CC'-U-R2-U-GK'

where the variables have the values described above. The composition has at least one parameter measured at 37°C, selected from the elastic modulus G' value of not less than about 50 PA, measured at a frequency of 1 Hz using a flat geometry 4 cm, and the kinematic viscosity of not less than approximately 2000 SP measured at shear rate 1 s-1. And the composition is sufficiently stable to enzymatic degradation observed when connecting composition is ri 37°C with the enzyme hyaluronidase in the amount of approximately 0.3% by weight, under conditions suitable for hyaluronidase reaction, the value of G' for the composition measured after 16 h the reaction is not less than approximately 5% of the value of G'measured at less than approximately 15 min from the beginning of the reaction. In another implementation, the value of G' for the composition measured after 16 h the reaction is at least about 50% of the value of G'measured at less than approximately 15 min from the beginning of the reaction.

As used here, "if necessary, increase tissue" means that the subject medical condition may benefit from the introduction of songs from the Ledger of the invention, for example a subject in need of treatment or correction of such conditions as wrinkles, furrows or creases and other wrinkles on the skin, usually on the forehead and around the eyes, nose and lips, correction or re-formation of soft tissue defects and tarabzouni scars. These conditions may be inherited or acquired as a result of aging, disease, injury, surgical complications, etc.

The term "subject" includes mammals such as humans, domestic animal (e.g. dogs, cats, birds and the like), farm animals (such as cows, pigs, horses, poultry and the like) and laboratory animals (e.g. rats, mice, Guinea pigs, birds etc). Most preferably the subject is a human who K.

As used here, "alkyl group" include, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, Needell, eicosyl, genacote, docosyl, tricosal, tetracosyl, pentasil and their isomeric forms. "Alkoxy group" is alcalina group connected through an oxygen atom, such as methoxy, ethoxy, propoxy, etc.

As used here, "alkenylamine groups are alkyl groups containing from 2 to 25 carbon atoms, which contain a double bond, such as vinyl, allyl, butenyl, pentenyl, hexenyl, octenyl, nonanal, decanal, undecanal, dodecanal, tridecanal, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, nonadecane, eicosanol, heneicosanol, docosanol, triazinyl, tetracosane, pentacosane and their isomeric forms.

As used here, "alkyline groups are alkyl groups comprising from 2 to 25 atoms containing a triple bond such as vinyl, allyl, butenyl, pentenyl, hexenyl, octenyl, nonini, decenyl, undecenyl, dodecenyl, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, nonadecane, eicosanol, heneicosanol, docosanol, triazinyl, tetralinyl, pentacosane and their isomeric forms.

The term "aryl", as PR is changing here toneset to phenyl, tollu, xiliu, the naphthyl biphenyl, triphenyl, etc. the Term "heteroaryl" refers to heteroaromatic groups, such as pyrrolyl, tieniu, furanyl, pyridinyl, oxazolyl, isooxazolyl, thiazolyl, isothiazoline, chinoline etc. "Kalkilya group is aryl group linked through alkyl group, for example benzyl, eternity etc.

As used here, "cycloalkyl" includes saturated ring of 3 to 8 carbon atoms, inclusive, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl etc. "Cycloalkylcarbonyl group" is cycloalkyl group linked through alkyl group, such as methylcyclopropyl etc.

The term "heterocyclyl" refers to cycloalkyl group, where one or more carbon atoms of the ring substituted by a heteroatom, such as airider, azetidin, pyrrolidyl, piperidyl, thiiranes, titanyl, tetrahydrothiophene, tetrahydrothiopyran, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl etc.

The term "cycloalkenyl" refers to cycloalkenyl groups having a double bond, for example cyclopropyl, cyclobutyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctyl etc.

The term "cycloalkenyl" refers to cycloalkenyl groups having a triple bond, such as Cyclops Pinel, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctyl etc.

The term "substituted", as used here, refers to a group in which one or more hydrogen atoms substituted with a chemical group that does not have an adverse effect on the preferred derivatization product. Representatives of such groups are halogen (e.g.,- F, -Cl, -Br, -I), amino, nitro, cyano, -HE, alkoxy, alkyl, aryl, amido, sulfamido, sulfate, sulfonate, phosphate, phosphonate, carboxylate, carboxamido etc.

Examples of carrying out the invention

Stitched SC receive the following examples by reacting bis-carbodiimide with unstitched hyaluronic acid in the specified proportions. Molecular weight unstitched Ledger used in these examples ranged from approximately 5×105Daltons to about 2×106Dalton, although the Ledger with a larger or smaller molecular weight can also be used. Applied Ledger received from the comb of a rooster or bacterial sources. All compounds were obtained from Sigma, St.Louis, MO in addition to the separately recorded cases.

Receiving MES-buffer (pH interval of 5.2 and 7.1)

For each MES-buffer, 2-[N-morpholino]econsultancy acid (MES hydrate) (14.6 g) was dissolved in 980 ml of sterile water. For each of the three solutions pH was adjusted to the desired value (of 5.5, 6.0 or 6.5 in), adding a 0.1 N NaOH, the Volume adjusted to 1 l, adding sterile water.

Phosphate buffers 1-5

For each individual phosphate buffer using 1 l of sterile water for dissolution specified in the Table 1 quantities of sodium phosphate dvenadcatiletnego (Na3PO4·12H2O), lidocaine hydrochloride (lidocaine ·HCl), sodium phosphate (disubstituted) (Na2HPO4) and phosphate one-deputizing water (NaH2PO4·H2O).

Table 1
Components of phosphate buffers 1-5
No. phosphate bufferNa3PO4·12H2Olidocaine ·HCINa2HPO4NaH2PO4·H2O
13.4 g, 9.0 mm---
23.4 g, 9.0 mm2.0 g ->0,20%--
33.4 g, 9.0 mm3.0 g ->0,30%--
42,48 g, 6.5 mm3.0 g ->0,30%0.36 g, 2.5 mm-
5-3.0 g ->0,30%1.42 g, 10.0 mm0.27 g, 2.0 mm

The method of examples 1-9

In each of the following examples, the reactants are used in amounts shown in table 2. Unstitched Ledger dissolved in 133,4 ml MES buffer with the specified pH and combined with the acetone solution of para-phenylene-bis(ethylcarbodiimide) (FBCCI) with a concentration of 15 mg/ml, receiving the specified ratio of molar equivalents (IEN %) and mol % between FBCCI and General Ledger. The reaction mixture is thoroughly mixed (using either a glass rod or the upper mechanical stirrer, for example, within 1 min, which leads to the formation of a white paste made of transparent reaction mixture and the mixture is left to stand at room temperature for about 72 hours. In the formed gel is blended sodium chloride (6.5 g to obtain the concentration of sodium chloride 5% by weight) and allowed to stand for 1 hour. Gel stitched CC precipitated by adding this mixture to approximately 1.2 l intensively mixed ethanol. The precipitate is collected and dried under reduced pressure to get stitched Ledger.

Table 2
components to get stitched CC in examples 1-9.
ExampleFBCCIAcetoneGKIENMol %pH
mgmmolMEQmlgmmol
11000,470,946,72to 4.9818,79,45,5
22000,941,8713,32to 4.9837,518,85,5
3267 1,252,512,62to 4.985025,05,5
44001,873,7426,72to 4.987537,55
54001,873,7426,72to 4.987537.55,5
64001,873,7426,72to 4.987537,56
74001,873,7426,72 to 4.987537,56,5
85342,5535,62to 4.9810050,05,5
96673,126,244,52to 4.9812562,55,5

Example 10: the Degree of crosslinking increases with increasing molar ratio in favor of the cross-linking agent

Crosslinking of hyaluronic acid para-phenylene-bis(ethylcarbodiimide) introduces phenylenebis chromophore that absorbs in the UV, product blending. By measuring the absorption cross-linked product in the UV can quantify the amount of UV chromophore and the degree of crosslinking, including the derived bis-carbodiimide as ligaments.

For solution preparation with a concentration of 0.1 mg/ml-weighted number (1-10 mg) of each of the staple products in examples 5, 8 and 9 separately dissolved in sufficient Koli is este solution of 5%sulfuric acid by heating at 70-75°C for 4 hours in a sealed container. Each diluted solution of 5%sulfuric acid to obtain a solution of each product with a concentration of 0.1 mg/ml Absorption in the ultraviolet (UV max) of such solutions is measured at 249 nm, shown in Figure 1 as a function of molar ratio of equivalents of bis-carbodiimide: GC %. Thus, visible correlation between the degree of crosslinking, including the derived bis-carbodiimide in the form of a ligament (as demonstrated by the values of the UV max), and the ratio of molar equivalents of bis-carbodiimide: SC in %.

Example 11: the Degree of crosslinking can be controlled via the pH.

For solution preparation with a concentration of 0.1 mg/ml-weighted number (1-10 mg) of each of the staple products in examples 5, 6 and 7 separately dissolved in sufficient quantity of a solution of 5%sulfuric acid by heating at 70-75°C for 4 hours in a sealed container, and then left to stand for 16 hours at room temperature. Such solutions are diluted with 5%sulfuric acid to obtain a solution of each product with a concentration of 0.1 mg/ml Absorption in the UV of such solutions is measured at 249 nm, shown in Figure 2.

You can see that the greatest degree of crosslinking, including the derived bis-carbodiimide in the form of bundles is achieved at pH 5.5 (composition according to example 5), which is greater than at pH 6.0 (composition of example 6), and these two more than at pH 6.5 (to the position from example 7).

Example 12: preparation of a gel from dehydrated crosslinked Ledger.

A portion of the dry residue stitched Ledger from example 5 grind in a cryogenic mill. The powder is suspended in dimethyl sulfoxide (DMSO) and the suspension stirred for 4-10 hours. The suspension is centrifuged and the DMSO is removed. Water-insoluble crosslinked Ledger resuspended in ethanol, stirred for several hours, and the ethanol removed (washing with ethanol, if necessary, can be repeated). After removal of ethanol, the solid is collected and dried under vacuum. Dry powder stitched CC suspended in phosphate buffer 4 for receiving the suspension 30 mg/ml in the form of a thick mass or paste. The osmolarity of the suspension is brought to 280-340 mOsm (milliosmoles) by adding sodium chloride and fill the syringe. The syringe autoclave at 120°C and a pressure of about 138 kPa (Pascal) (20 pounds/square inch) for 25 min and cooled after sterilization with cold water.

Example 13: a description of the elastic modulus G' of the gel hydrated Ledger.

Rheological behavior (including the elastic modulus G'of the gel from example 12 was evaluated using a Rheometer AR 1000 (TA Instrument, New Castle, DE). The measurement conditions include a temperature equal to 37°C, the surface geometry representing a flat plate, a gap of 200 μm (micrometers) and a frequency of 1 Hz. Measured in these conditions, the elastic modulus G' of the gel obtained is about example 12, was 1076 PA (Pascal).

Example 14: the characteristic strength of the extrusion (extrusion) of hydrated gels Ledger

The force required for extrusion of the gel can be characterized by loading the gel in a glass syringe 1 ml with an inner diameter of 0,635 cm, equipped with a needle 30-th size (internal diameter of approximately 150 μm, the cross-sectional area 0,0177 mm2). The force required for extrusion of the gel through a needle at a speed of 4 ml/h, was measured by placing the syringe in a syringe pump connected to the system of measurement of force (Load Cell SLB-50, Transducer Techniques, CA). At approximately 25°C and the speed of extrusion of 4 ml/hour measured extrusion force comprised 10.7 N (Newton) (2.4 lbs).

Example 15: Characteristics of stability of hydrated gels Ledger to the enzymatic hydrolysis.

Durability stitched CC enzymatic hydrolysis is measured by test in vitro, combining the gel with the measured amount of enzyme hyaluronidase and registering the modulus of elasticity as a function of time.

The gel of example 12 (0.75 g), placed in the flask, add a solution of hyaluronidase from bull testes (15 μl of a solution of hyaluronidase 0.15 mg/ml 1.9 mm phosphate buffer in saline, approximately 2.5 units) and mix thoroughly for 1 min with a Mixture fill the syringe and centrifuged for 1 min at 1500 rpm for compa the Oia air bubbles. 0.35 g of this mixture is placed on the plate of the rheometer. Data collection starts in 15 min after addition of the enzyme. The elastic modulus G' of the gel is recorded at intervals of 10 min for 16 hours at 37°C on a flat plate with a gap of 200 μm and a frequency of 1 Hz.

The rate of decrease in the elastic modulus ΔG'/Δt, i.e. the slope of the plot of G' from time to time, calculated by dividing the ΔG' on the time interval. This speed may represent the sensitivity of the product to the enzymatic hydrolysis. When comparing the stability of two compositions composition with greater stability, better support for modulus of elasticity than a composition with less stability. In this example, the average loss rate G' of the gel is about -5 PA/h. Thus, the composition of the sample can be considered stable in the conditions of the test. Experienced in this field specialist will understand that the compositions are stable in such conditions, you can expect good stability in vivo.

Example 16: Effect of particle size on the rheological properties of the gel.

Dry the precipitate stitched Ledger from example 5 grind in a cryogenic mill and washed with DMSO and ethanol as described in example 12. After removal of ethanol, the solid is collected and dried under vacuum. Dry powder stitched CC fractionary at an average diameter of five sieves: 25 μm, 75 m is m, 125 μm, 180 μm and 250 μm, placed in a stack (one above the other). Collect five fractions by average diameter: 0-25 μm, 25-75 μm, 75-125 microns, 125-180 180-250 microns and microns. Each fraction is suspended in phosphate buffer 5 to obtain a suspension of 32 mg/ml Osmolarity of the suspension is brought to 280-340 mOsm (milliosmoles) and each suspension is loaded into a syringe. Syringes autoclave at 120°C and a pressure of about 138 kPa (Pascal) (20 pounds/square Inch) for about 45 min and then cooled with cold water.

The figure 3 shows the elastic modulus (G') for each gel, measured in conditions described in example 12. As can be seen in figure 3, the rheological properties of viscoelastic gels depend on the distribution of the average particle diameter and composition of the gel.

The figure 4 shows the force required for extrusion of these gels through a needle 30-th size according to the method described in example 12. As can be seen in figure 4, the force of extrusion such viscoelastic gels depends on the distribution of the mean diameter of the particles and gels.

Example 17. Combining fractions according to particle size to obtain the special rheological properties.

Dry besieged stitched Ledger from example 5 grind and washed as described in example 12. A portion of the dry powder stitched CC then sieved through a sieve of 250 μm. All particles of average diameter less than 250 μm collect suspendered in phosphate buffer 4 for receiving the suspension 32 mg/ml

Two batches of particles with an average diameter 250-125 μm and less than 125 microns are collected. These fractions are mixed in a ratio of 1:1 by weight and suspended in phosphate buffer 4 for receiving the suspension 32 mg/ml Osmolarity of each suspension was adjusted to 280-340 mOsm (milliosmoles) and loaded into the syringe. Syringes autoclave at 120°C and a pressure of about 138 kPa (Pascal) (20 pounds/square inch) for about 45 min and then cooled with cold water.

The elastic modulus (G') for these gels is measured in conditions described in example 12. A sample of the gel prepared from particles with an average diameter of less than 250 μm, has an initial G' 600 PA; sample gel prepared from particles with an average diameter 250-125 μm and 125-0 μm in the ratio of 1:1, it has the initial G' 450 PA.

Example 18: the Modulus of elasticity as a function of the degree of crosslinking.

A portion of dry precipitated stitched CC of each of examples 5 and 6 milled and washed as described in example 12. Each portion was then sieved through a sieve of 250 μm. Particles with an average diameter of less than 250 microns are collected and suspended in phosphate buffer 4 for receiving the suspension 32 mg/ml

The osmolarity of each suspension was adjusted to 280-340 mOsm (milliosmoles) and loaded into the syringe. Syringes autoclave at 120°C and a pressure of about 138 kPa (Pascal) (20 pounds/square inch) for about 20 min and then cooled in cold water is th.

The elastic modulus (G') for these gels is measured using the measurement conditions described in example 12. A sample of the gel prepared from the composition of example 5 had an initial G' 700 PA; a sample of the gel prepared from cross-linked composition of example 6 had the initial G' 450 PA. It is seen that the composition with a greater degree of crosslinking by measuring the degree of crosslinking in the UV, i.e. the composition of example 5, also has a large modulus of elasticity.

Example 19: ΔG'/Δt of the invention is superior to that of competing products.

Stitched CC obtained in example 5 and treated according to example 12, turns into a gel with the initial G' 450 PA. Evaluated the sustainability of this product and three competing products for building tissues of Restylane®, Perlane® (both from Q-Med, Uppsala, Sweden) and Hylaform® (Genzyme, Cambridge, MA) to digestion by hyaluronidase. The hyaluronidase solution (15 μl of a solution of hyaluronidase 0.15 mg/ml 1.9 mm phosphate buffer, ≈2.5 units) are added to 0.75 g of each product and mix thoroughly. Each mixture was loaded into a syringe and centrifuged for 1 min at 1500 rpm to remove air bubbles. Then 0.35 g of each mixture was placed on the plate of the rheometer. To collect data beginning after 15 min from the moment of addition of the enzyme. The elastic modulus G' of the gel recorded at intervals of 10 min for 16 hours at 37°C on a flat plate, for which the PR is 200 μm and a frequency of 1 Hz.

The figure 5 shows graphs of G' with time for each product. As can be seen, the loss rate representing the exposure of the product of the enzymatic hydrolysis, much less the compositions of the invention than three competing songs.

Example 20: ΔG'/Δt composition against hyaluronidase does not depend on G'.

The figure 6 shows ΔG'/Δt over a period of 16 hours for a set of compositions with crosslinked Ledger with the initial values of modulus of elasticity between about 200 and about 1200 PA. Compositions were obtained and measurements were carried out on the above described examples. As can be seen, the degradation of the compositions with crosslinked ha, largely depends on the initial elastic modulus G' for such a period of 16 hours.

Example 21: Synergistic action of lidocaine on the rheological properties of the gel. Lidocaine may have a synergistic effect and increase the initial elastic modulus G' of the gel compared to the identical besides the compositions obtained in the buffer without lidocaine. Stitched CC of example 5 was treated as described in example 12, using three different phosphate buffers 1 (without lidocaine), 2 (0.2% lidocaine) and 3 (0.3% lidocaine). Prepared gels with a concentration of 32 mg/ml and measured the elastic modulus G' and the degradation profiles ΔG'/Δt of each according to the method described in example 12. In figure 7 it is shown that the composition with lidocai the ohms have a substantially higher modulus of elasticity during the test. Thus, stitched CC with lidocaine may have a high biostability and in some cases to have a synergistic effect with the increase of G'.

Example 22: the Invention is effective for increasing tissue.

Product stapling Ledger used in this study were prepared according to the method described in example 14. In the study of intradermal (interdermal) injection in Guinea pigs as a model stitched product Ledger and the control substance (Zyderm™II, Collagen, Palo Alto, Ca) was injected intradermally (intradermal). Each injection site (6 at the time the sample) measured the height and diameter after 2, 4, 8 and 12 weeks. The samples were taken and cultured in an artificial environment for histological evaluation for each time interval.

The study included twelve Guinea pigs, distributed on two for each time interval of the experience of the 2, 4, 8, 12, 18 and 24 weeks.

With the right and left sides of animals sheared wool not less than an hour before a dose. Each animal received six intradermal injections three per side. The test substance and the reference substance was distributed randomly on each animal. Both substances were dosaged 0.2 cm3on the plot. Each plot is labelled non-toxic marker, the injection sites were approximately 2 cm from each other. the same height and diameter of each plot was measured through each time interval. Measurements immediately after the injection attributed score (degree) "0". The increase in height and in diameter indicates Eden (edema).

At each interval the complete experience was slaughtered three animals, sheared fur, measured the injection site, deleted the site and fixed it in 10%neutral buffered formalin. Then fabric zaplavlyali in blocks, made the cut and were stained with hematoxylin and eosin (H&e) and subjected to examination by a qualified pathologist. The specimens were examined for the presence of introduced substances and any tissue reaction.

In the first week of experiments points (degree) erythema were minimal (weak) and was equally distributed between the sites of injection with the test substance and Ziderman™II. Points (degree) oedema were irregular and equally distributed between the two substances. In the second week points (degree) erythema were similar to the first week. Points (degree) oedema have been reduced, falling to zero. The measurement sites of injection on the 4th week were equal to the measurements obtained immediately after injection.

The amount of one HUNDRED (tissue growth) did not change from 2 weeks to 4 weeks. At 8-week follow-up almost all of the sites with the test substance remained dimensions of height and diameter. On the contrary, almost all control plots were not measured. At 12-week follow-sections tested with the m substance oposals and distributed in width. However, in areas of the dermis with higher density tissue test substance did not spread to the same extent as in areas with less. Fibroblasts and adipose tissue penetrated the test substance approximately the same density as the tissue adjacent to the injection site. The control substance is not identified on any of the sites of injection.

Microscopic examination did not show any cellular response to the test substance and only a very small infiltration of macrophages in the case of the control of matter on the 2nd week and 4th week. On the 8th and 12th weeks were observed cellular responses to any material.

2, 4, 8 and 12 weeks of the sites with the test substance were deprived of any tissue response, confirming the biocompatibility of this drug. After the injection of the test substance, apparently, is integrated in the stromal elements of the dermis.

The dimensions in the height and diameter did not change between 2 and 4 weeks. Apparently, the 8 week one HUNDRED and spread laterally (sideways) in the dermal layers, but still kept its volume. In contrast, the control substance appears in the form of a homogeneous ball of material pressing on the underlying dermis. Small infiltration with macrophages observed for the control substances for 4 week, 8 week, most of the injection of rassas is provided with the injection site.

The stability of the test substance in vivo at 8 and 12 weeks compared with the control substance indicates that this product may be a long life in clinical applications. These results support the in vitro data described above, demonstrating resistance to collapse under the action of hyaluronidase in vivo.

Although the invention, in particular, shown and described above with reference to selected exercise, anyone experienced in this field specialist will be understood that various changes in form and details may be done without departing from the scope and field of application of the invention defined in the attached claims.

1. Injectable composition of hyaluronic acid to increase tissue from a subject in need of building fabric, or for delivery of medicines containing particles of gel crosslinked water-insoluble hydrated ON where TO include ligaments, represented by the following structural formula
HA'-U-R2-U-HA',
in which
each group' means the same or another molecule sewn ON';
each U is independently denotes optionally substituted O-utilization or N-allocatio; and
R2represents an optionally substituted alkyl, alkenyl, quinil, alkoxy, cycloalkyl, cycloalkenyl, cycloalkenyl, aryl, heteroaryl, heterocyclic the ski radical, cycloaliphatic alkyl, aralkyl, heteroalkyl or geterotsiklicheskie, and
particles have a distribution of the average diameter selected from the group consisting of particles with a distribution according to the average diameter selected from the group consisting of hydrated particles with an average diameter of approximately between 20 and 1000 microns and digidrirovanny particles with an average diameter of between approximately 10 and 500 μm,
the injectable composition suitable for increasing tissue from a subject in need of building up tissue, and/or to ensure the effective introduction and/or discharge medications.

2. Composition under item 1, in which the particles include at least one bioactive agent selected from the group consisting of cells, genes, proteins, antibodies, peptides and pharmaceutical substances.

3. Composition ON p. 2, where the bioactive agent comprises an anesthetic.

4. The composition according to p. 3, where the bioactive agent means lidocaine, mepivacaine, prilocaine, bupivacaine, cocaine, procaine, chloroquin or tetracaine, or salt, or MES.

5. Composition ON p. 4, where the bioactive agent means lidocaine HCl.

6. Composition under item 5, in which the anesthetic is included in the particles ON the gel in the amount of at least about 0.1% by weight of particles ON the gel, where the value of the elastic modulus G' during storage DL the song which includes a local anesthetic is at least about 110% of the value of G'measured for a composition that does not contain a local anesthetic, when changing at 37°C at a frequency of 1 Hz, using a sample with a flat geometry and size 4

7. Composition ON p. 6, in which the value of G' for the composition, which includes a local anesthetic is at least approximately 150% of the value of G' for the composition that does not contain a local anesthetic.

8. Composition under item 5, in which:
a) the composition has at least one parameter measured at 37°C and selected from the modulus G' during storage equal to at least about 50 PA, when measured at a frequency of 1 Hz, using a sample with a flat geometry and a size of 4 cm, and the kinematic viscosity, equal to at least approximately 20,000 centipoise, measured at shear rate 1 s-1;
b) the composition is sufficiently stable to enzymatic degradation, so when combining composition at 37°C with the enzyme hyaluronidase in the amount of about 0.3 wt.% under conditions suitable for interaction with hyaluronidase, the value of G' composition measured after 16 hours of interaction is at least about 5% of the value of G'measured at least approximately 15 min interaction is the major.

9. Composition ON p. 8, in which the value of G' for the composition, measured after 16 h of interaction is at least about 50% of the value of G'measured less than approximately 15 min of interaction.

10. Composition under item 1, in which the composition has at least one parameter measured at 37°C, selected from the elastic modulus G' during storage, is approximately equal to at least 50 PA measured at a frequency of 1 Hz using a flat geometry of the sample 4 cm, and the kinematic viscosity approximately equal to at least 20000 centipoise measured at shear rate 1 s-1.

11. Composition under item 9, in which after combining the composition at 37°C with the enzyme hyaluronidase in the amount of about 0.3 wt.% under conditions suitable for interaction with hyaluronidase, the value of the modulus G' during storage of the composition, measured after 16 h of interaction is at least about 5% of the value of G'measured approximately less than 15 min of interaction.

12. Method of increasing tissue in a subject in need of building fabric, including the steps:
a) inserting a needle to the subject in the place where you want to build tissue in the subject, and a needle connected to a syringe filled with composition sewn ON, which includes particles of gel crosslinked, insoluble in the e, hydrated gel particles, where includes ligaments, represented by the following structural formula:
HA'-U-R2-U-HA',
in which
each' is the same or a different molecule sewn ON;
each U is independently denotes optionally substituted O-utilization or N-allocatio; and
R2means optionally substituted alkyl, alkenyl, quinil, alkoxy, cycloalkyl, cycloalkenyl, cycloalkenyl, aryl, heteroaryl, heterocyclic radical, cycloaliphatic alkyl, aralkyl, heteroalkyl or heteroseksualci; and
the particles are not soluble in water and have the distribution of the average diameter selected from the group consisting of hydrated particles of average diameter between about 20 and 1000 microns, and digidrirovanny particle average diameter between about 10 and 500 microns; and
b) application to the syringe such force that at least part of the song was introduced ON the subject.

13. The method according to p. 12, in which the subject is the man.

14. The method according to p. 13, wherein the particles include at least one bioactive agent selected from the group consisting of cells, genes, proteins, antibodies, peptides and pharmaceutical substances.

15. The method according to p. 14, in which the bioactive agent comprises a local anesthetic.

16. The method according to p. 15, in which the bioactive agent is in minicamera, lidocaine, mepivacaine, prilocaine, bupivacaine, cocaine, procaine, chloroquin or tetracaine, or salt, or MES.

17. The method according to p. 16, in which the bioactive agent means lidocaine HCl.

18. The method according to p. 12, in which the composition has at least one parameter measured at 37°C, selected from the modulus G' during storage equal to at least approximately 50 PA measured at a frequency of 1 Hz using a flat geometry of the sample 4 cm, and kinematic viscosity, equal to at least approximately 20,000 centipoise, measured at shear rate 1 s-1.

19. The method of obtaining injectable compositions of hyaluronic acid to increase tissue from a subject in need of building fabric, or for drug delivery, comprising the steps:
a) the formation of particles insoluble in water, dehydrated, sewn ON by:
i) stapling predecessor sewn ON using bis-carbodiimide in the presence of buffer at pH value between approximately 4 and 8, and formed sewn ON includes knitting, represented by the following structural formula
HA'-U-R2-U-HA',
in which
each' is the same or a different molecule sewn ON';
each U is independently denotes optionally substituted O-utilization or N-allocatio and
R2means the optional Sames the config alkyl, alkenyl, quinil, alkoxy, cycloalkyl, cycloalkenyl, cycloalkenyl, aryl, heteroaryl, heterocyclic radical, cycloaliphatic alkyl, aralkyl, heteroalkyl or heteroseksualci; and
ii) dehydration sewn ON with getting dehydrated sewn ON.
b) grinding dehydrated sewn ON with the formation of insoluble particles in water, sewn ON;
c) separating insoluble in water, digidrirovanny particle mean diameter and selecting a subset of the particle mean diameter; and
d) hydration subset digidrirovanny particles using a physiologically compatible aqueous solution to obtain a composition.

20. The method according to p. 19, in which digidrirovannye particles hydratious in the presence of a physiologically acceptable solution at conditions including a temperature of at least about 100°C., a pressure of at least approximately 120 kPa and a duration of at least approximately 15 minutes

21. The method according to p. 19, in which the physiologically compatible aqueous solution contains a bioactive agent.

22. The method according to p. 21, where the bioactive agent is lidocaine, mepivacaine, prilocaine, bupivacaine, cocaine, procaine, chloroquin or tetracaine, or salt, or MES.

23. The method according to p. 22, where the bioactive agent is lidocaine HCl.

24. The method according to p. 19, in which the pH of the pillar is t about 5.5.

25. The method according to p. 19, in which the buffer contains at least one buffering agent selected from the group consisting of 2-(N-morpholino)econsultancy acid; 2,2-bis(hydroxymethyl)-2,2',2"-microtiterplate/succinic acid;
KH2PO4; N-Tris(hydroxymethyl)-2-aminoethanesulfonic acid; triethanolamine; diethylbarbituric; Tris(hydroxymethyl)aminoethane; N-Tris(hydroxy)methylglycine and N,N-bis(2-hydroxyethyl)glycine.

26. The method according to p. 19, in which bis-carbodiimide is at least one compound selected from the group consisting of 1,6-hexamethylene-bis(ethylcarbodiimide), 1,8-octamethylene-bis(ethylcarbodiimide), 1,10-decametre-bis(ethylcarbodiimide), 1,12-dodecamethyl-bis(ethylcarbodiimide), polyethylene glycol-bis(propyl(ethylcarbodiimide)), 2,2'-Dityatin-bis(ethylcarbodiimide), 1,1'-dithio-para-phenylene-bis(ethylcarbodiimide), para-phenylene-bis(ethylcarbodiimide) and 1,1'-dithio-meta-phenylene-bis(ethylcarbodiimide).

27. The method according to p. 19, in which a subset digidrirovanny particles selected in stage (C), consists of digidrirovanny particle average diameter between about 10 and 500 microns, and hydrated particles of average diameter between about 20 and 1000 microns.



 

Same patents:

FIELD: chemistry.

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

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

3 cl, 3 ex

FIELD: medicine.

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

EFFECT: implants of simplified injection.

14 cl, 4 ex

The invention relates to medicine, namely to a restorative or cosmetic surgery and aesthetic dermatology

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: medicine.

SUBSTANCE: are described medical implants which release anthracycline, fluoropyrimidine, antagonist of folic acid, podophyllotoxin, camptotecine, hydroxyurea or platinum complex.

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83 cl, 1 dwg, 2 tbl, 25 ex

FIELD: medicine.

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EFFECT: increase of biological integration of polyester prostheses.

15 cl, 2 tbl

FIELD: medicine.

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EFFECT: reliable fixation of antibiotic solution within porous implant and arrangement of favourable conditions for haemostasis in operative wound.

3 cl, 1 ex

FIELD: medicine.

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27 cl, 1 dwg

FIELD: medicine.

SUBSTANCE: method has stages for manufacturing medical device and introducing effective dose of Linezolid.

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17 dwg, 11 dwg, 6 tbl

FIELD: medical equipment.

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EFFECT: higher reliability of attachment of implant.

21 cl, 3 tbl, 10 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: the present innovation deals with a medicinal prosthesis that contains metallic material, such as titanium or its alloy, in which surface parts of metallic material area covered with the layer of the corresponding hydroxide material, such as titanium hydroxide. Preferably, hydroxide layer contains one or more biomolecular substances being connected with it. Also, the innovation in question refers to electrolytic process for obtaining a medicinal prosthesis. Metallic prostheses are of improved biological compatibility.

EFFECT: higher efficiency.

24 cl, 8 ex, 3 tbl

FIELD: medicine.

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EFFECT: enhanced effectiveness of treatment in cognitive-mnestic function disorder cases.

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Transplant mixture // 2301684

FIELD: medicine.

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EFFECT: enhanced effectiveness of treatment; no clamps required; reliably and tightly closed bone tissue defect; accelerated regenerate reorganization; improved antiseptic and immunomodulating action.

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: medicine.

SUBSTANCE: method of antibiotics fixation within porous implants is described. Result of method application lies in possibility of reliable fixation of antibiotic solution within porous implant and arrangement of favourable conditions for haemostasis in operative wound due to application of 10% gelatine solution as antibiotic carrier. Specified result is achieved by filling porous implants with antibiotic solution in liquid gel. For this purpose implant is dipped in solution by 3/4. Filling occurs under the influence of capillary forces. After solution cooled to form dense gel, antibiotic is fixed in implant pores and gradually released after installation to bone defect area.

EFFECT: reliable fixation of antibiotic solution within porous implant and arrangement of favourable conditions for haemostasis in operative wound.

3 cl, 1 ex

FIELD: medicine-destination polymers.

SUBSTANCE: invention relates to biologically stable hydrogels to be employed as endoprosthesis consisting essentially of following components: polyacrylamide including acrylamide, crosslinked methylene-bis-acrylamide, wherein acrylamide and methylene-bis-acrylamide are linked at molar ratio from 150:1 to 1000:1. Hydrogel is rinsed with water or physiologic solution so that it contains about 0.5-3.5% polyacrylamide and less than 50 ppm acrylamide and methylene-bis-acrylamide monomers, while modulus of elasticity of hydrogel is approximately 10 to 700 Pa and its complex viscosity about 2 to 90 Pa*sec. Rinsing stage allows removal of nearly all amounts (even trace amounts) of above-indicated monomers resulting in lower toxicity and higher stability of hydrogel. Biologically stable hydrogel is used as injectable prosthesis to fill soft tissues and also to treat or prevent urinary incontinence or anal incontinence. Hydrogel, obtained in a few stages including combining acrylamide and methylene-bis-acrylamide, initiating radical polymerization, and rinsing with apyrogenic water or physiologic solution, is also useful in treatment or prevention of bladder-ureter reflux in mammalians. In all these cases biologically stable hydrogels contain between 0.5 and 25% polyacrylamide.

EFFECT: enlarged resource for manufacturing endoprostheses.

10 cl, 3 dwg, 7 tbl

The invention relates to medicine, in particular to plastic surgery
The invention relates to medicine, namely to a method for producing compositions for injection, for use in reconstructive and cosmetic surgery

The invention relates to medicine, namely to molecular-linked gel containing a variety of biological and non-biological polymers such as proteins, polysaccharides and synthetic polymers

The invention relates to a formulation and method for producing a biocompatible hydrogel based on cross-linked copolymer of acrylamide with cross-linking agents that can be used as a material for medical purposes, for example:

- when the endoprosthesis through targeted injections hydrogel for plastics soft tissues of the face, breast, penis, calf muscles, vocal cords and other tissues, the density of which corresponds to the density of the hydrogel;

- as a filler in the manufacture of implants, including implants breast;

- as a depot for drugs with long-term medication, such as tumors or abscesses;

- as a carrier for culturing human cells and animals with subsequent implantation of hydrogel containing cells in the mammalian organism
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